Chronic Rotenone Research Articles

High Yield of Functional Dopamine-like Neurons Obtained in NeuroForsk 2.0 Medium to Study Acute and Chronic Rotenone Effects on Oxidative Stress, Autophagy, and Apoptosis.

Several efforts to develop new protocols to differentiate in in vitro human mesenchymal stromal cells (hMSCs) into dopamine (DA) neurons have been reported. We have formulated NeuroForsk 2.0 medium containing fibroblast growth factor type beta (FGFb), brain-derived neurotrophic factor (BDNF), melatonin, purmorphamine, and forskolin. We report for the first time that menstrual stromal cells (MenSCs) cultured in NeuroForsk 2.0 medium for 7 days transdifferentiated into DA-like neurons (DALNs) expressing specific DA lineage markers tyrosine hydroxylase-positive cells (TH+) and DA transporter-positive (DAT+) cells and were responsive to DA-induced transient Ca2+ influx. To test the usefulness of this medium, DALNs were exposed to rotenone (ROT), a naturally occurring organic neurotoxin used extensively to chemically induce an in vitro model of Parkinson's disease (PD), which is a movement disorder characterized by the specific loss of DA neurons. We wanted to determine whether ROT induces apoptotic cell death and autophagy pathway under acute or chronic conditions in DALNs. Here, we report that acute ROT exposure induced several molecular changes in DALNS. ROT induced a loss of mitochondrial membrane potential (ΔΨm), high expression of parkin (PRKN), and high colocalization of dynamin-related protein 1 (DRP1) with the mitochondrial translocase of the outer membrane of mitochondria 20 (TOMM20) protein. Acute ROT also induced the appearance of DJ-1Cys106-SO3, as evidenced by the generation of H2O2 and oxidative stress (OS) damage. Remarkably, ROT triggered the phosphorylation of leucine-rich repeat kinase 2 (LRRK2) at residue Ser935 and phosphorylation of α-Syn at residue Ser129, a pathological indicator. ROT induced the accumulation of lipidated microtubule-associated protein 1B-light chain 3 (LC3B), a highly specific marker of autophagosomes. Finally, ROT induced cleaved caspase 3 (CC3), a marker of activated caspase 3 (CASP3) in apoptotic DALNs compared to untreated DANLs. However, the chronic condition was better at inducing the accumulation of lysosomes than the acute condition. Importantly, the inhibitor of the LRRK2 kinase PF-06447475 (PF-475) almost completely blunted ROT-induced apoptosis and reduced ROT-induced accumulation of lysosomes in both acute and chronic conditions in DALNs. Our data suggest that LRRK2 kinase regulated both apoptotic cell death and autophagy in DALNs under OS. Given that defects in mitochondrial complex I activity are commonly observed in PD, ROT works well as a chemical model of PD in both acute and chronic conditions. Therefore, prevention and treatment therapy should be guided to relieve DALNs from mitochondrial damage and OS, two of the most important triggers in the apoptotic cell death of DALNs.

Inhibition of carnitine palmitoyl-transferase 1 is a potential target in a mouse model of Parkinson’s disease

Glucose metabolism is dysregulated in Parkinson’s disease (PD) causing a shift toward the metabolism of lipids. Carnitine palmitoyl-transferase 1A (CPT1A) regulates the key step in the metabolism of long-chain fatty acids. The aim of this study is to evaluate the effect of downregulating CPT1, either genetically with a Cpt1a P479L mutation or medicinally on PD using chronic rotenone mouse models using C57Bl/6J and Park2 knockout mice. We show that Cpt1a P479L mutant mice are resistant to rotenone-induced PD, and that inhibition of CPT1 is capable of restoring neurological function, normal glucose metabolism, and alleviate markers of PD in the midbrain. Furthermore, we show that downregulation of lipid metabolism via CPT1 alleviates pathological motor and non-motor behavior, oxidative stress, and disrupted glucose homeostasis in Park2 knockout mice. Finally, we confirm that rotenone induces gut dysbiosis in C57Bl/6J and, for the first time, in Park2 knockout mice. We show that this dysbiosis is alleviated by the downregulation of the lipid metabolism via CPT1.

C/EBPβ/AEP is age-dependently activated in Parkinson’s disease and mediates α-synuclein in the gut and brain

Parkinson’s disease (PD) is the most common neurodegenerative motor disorder, and its pathologic hallmarks include extensive dopaminergic neuronal degeneration in the Substantia nigra associated with Lewy bodies, predominantly consisting of phosphorylated and truncated α-Synuclein (α-Syn). Asparagine endopeptidase (AEP) cleaves human α-Syn at N103 residue and promotes its aggregation, contributing to PD pathogenesis. However, how AEP mediates Lewy body pathologies during aging and elicits PD onset remains incompletely understood. Knockout of AEP or C/EBPβ from α-SNCA mice, and their chronic rotenone exposure models were used, and the mechanism of α-Syn from the gut that spread to the brain was observed. Here we report that C/EBPβ/AEP pathway, aggravated by oxidative stress, is age-dependently activated and cleaves α-Syn N103 and regulates Lewy body-like pathologies spreading from the gut into the brain in human α-SNCA transgenic mice. Deletion of C/EBPβ or AEP substantially diminished the oxidative stress, neuro-inflammation, and PD pathologies, attenuating motor dysfunctions in aged α-SNCA mice. Noticeably, PD pathologies initiate in the gut and progressively spread into the brain. Chronic gastric exposure to a low dose of rotenone initiates Lewy body-like pathologies in the gut that propagate into the brain in a C/EBPβ/AEP-dependent manner. Hence, our studies demonstrate that C/EBPβ/AEP pathway is critical for mediating Lewy body pathology progression in PD.

AMPK-dependent autophagy activation and alpha-Synuclein clearance: a putative mechanism behind alpha-mangostin's neuroprotection in a rotenone-induced mouse model of Parkinson's disease.

Alpha-Synuclein (α-Syn) accumulation is central to the pathogenesis of Parkinson's disease (PD), hence the quest for finding potential therapeutics that may promote the α-Syn clearance is the need of the hour. To this, activation of the evolutionarily conserved protein and key regulator of the autophagy, 5'AMP-activated protein kinase (AMPK) is well-known to induce autophagy and subsequently the clearance of α-Syn aggregates. Alpha-mangostin (AM) a polyphenolic xanthone obtained from Garcinia Mangostana L. was previously reported to activate AMPK-dependent autophagy in various pre-clinical cancer models. However, no studies evidenced the effect of AM on AMPK-dependent autophagy activation in the PD. Therefore, the present study aimed to investigate the neuroprotective activity of AM in the chronic rotenone mouse model of PD against rotenone-induced α-Syn accumulation and to dissect molecular mechanisms underlying the observed neuroprotection. The findings showed that AM exerts neuroprotection against rotenone-induced α-Syn accumulation in the striatum and cortex by activating AMPK, upregulating autophagy (LC3II/I, Beclin-1), and lysosomal (TFEB) markers. Of note, an in-vitro study utilizing rat pheochromocytoma cells verified that AM conferred the neuroprotection only through AMPK activation, as the presence of inhibitors of AMPK (dorsomorphin) and autophagy (3-methyl adenine) failed to mitigate rotenone-induced α-Syn accumulation. Moreover, AM also counteracted rotenone-induced behavioral deficits, oxidative stress, and degeneration of nigro-striatal dopaminergic neurons. In conclusion, AM provided neuroprotection by ameliorating the rotenone-induced α-Syn accumulation through AMPK-dependent autophagy activation and it can be considered as a therapeutic agent which might be having a higher translational value in the treatment of PD.

Neurodegeneration in the centrally-projecting Edinger\u2013Westphal nucleus contributes to the non-motor symptoms of Parkinson\u2019s disease in the rat

BackgroundThe neuropathological background of major depression and anxiety as non-motor symptoms of Parkinson’s disease is much less understood than classical motor symptoms. Although, neurodegeneration of the Edinger–Westphal nucleus in human Parkinson’s disease is a known phenomenon, its possible significance in mood status has never been elucidated. In this work we aimed at investigating whether neuron loss and alpha-synuclein accumulation in the urocortin 1 containing (UCN1) cells of the centrally-projecting Edinger–Westphal (EWcp) nucleus is associated with anxiety and depression-like state in the rat.MethodsSystemic chronic rotenone administration as well as targeted leptin–saporin-induced lesions of EWcp/UCN1 neurons were conducted. Rotarod, open field and sucrose preference tests were performed to assess motor performance and mood status. Multiple immunofluorescence combined with RNAscope were used to reveal the functional–morphological changes. Two-sample Student’s t test, Spearman’s rank correlation analysis and Mann–Whitney U tests were used for statistics.ResultsIn the rotenone model, besides motor deficit, an anxious and depression-like phenotype was detected. Well-comparable neuron loss, cytoplasmic alpha-synuclein accumulation as well as astro- and microglial activation were observed both in the substantia nigra pars compacta and EWcp. Occasionally, UCN1-immunoreactive neuronal debris was observed in phagocytotic microglia. UCN1 peptide content of viable EWcp cells correlated with dopaminergic substantia nigra cell count. Importantly, other mood status-related dopaminergic (ventral tegmental area), serotonergic (dorsal and median raphe) and noradrenergic (locus ceruleus and A5 area) brainstem centers did not show remarkable morphological changes. Targeted partial selective EWcp/UCN1 neuron ablation induced similar mood status without motor symptoms.ConclusionsOur findings collectively suggest that neurodegeneration of urocortinergic EWcp contributes to the mood-related non-motor symptoms in toxic models of Parkinson’s disease in the rat.

Protective effect of alpha mangostin on rotenone induced toxicity in rat model of Parkinson’s disease

Parkinson’s disease (PD) is a progressive, late-onset, and degenerative disorder that affects the central nervous system with an unknown etiology. Due to its incredible complexity in disease nature, many of the existing treatment approaches show a vain recovery in Parkinson’s patients. Therefore, an in search of disease-modifying therapeutics for an effective recovery is essential. Alpha mangostin is an important polyphenolic xanthone reported for its neuroprotective effect against rotenone-induced α-synuclein aggregation and loss of tyrosine hydroxylase positive (TH+)-neurons in SH-SY5Y cells. Hence, the current study aims to test its protective effect in managing the in-vivo rat model of PD. To justify this aim, adult male Sprague Dawley rats (250 ± 20 g) were subjected to chronic treatment of rotenone (2 mg/kg/day, s.c.) for 21 days. In parallel alpha mangostin treatment (10 mg/kg, i.p) was administered along with rotenone for 21 days. Chronic rotenone treatment for 21 days increased lipid peroxidation, nitrite concentration, and decreased glutathione levels. Further, depletion of TH+-dopaminergic neuron expression in substantia nigra pars compacta (SNc), and the development of motor and behavioral deficits in rotenone treated animals like cognitive impairment, muscle incoordination, and neuromuscular weakness were observed. Moreover, western blot studies ascertained the reduced normal alpha-synuclein levels and increased phosphorylated α-synuclein levels in comparison to the vehicle-treated group. Treatment with alpha mangostin significantly restored the locomotor activity, memory deficits, and improved the levels of antioxidant enzymes. It also significantly reduced the levels of phosphorylated α-synuclein which in turn gave protection against TH+-dopaminergic neuronal loss in SNc, suggesting it’s anti-oxidant and anti-aggregatory potential against α-synuclein. In conclusion through our current results, we could suggest that alpha mangostin has a potential neuroprotective effect against rotenone-induced PD and might be used as a neuroprotective agent. Further mechanistic studies on preclinical and clinical levels are required to be conducted with alpha mangostin to avail and foresee it as a potential agent in the treatment and management of PD.

Phenothiazine normalizes the NADH/NAD+ ratio, maintains mitochondrial integrity and protects the nigrostriatal dopamine system in a chronic rotenone model of Parkinson's disease

Impaired mitochondrial function has been associated with the etiopathogenesis of Parkinson's disease (PD). Sustained inhibition of complex I produces mitochondrial dysfunction, which is related to oxidative injury and nigrostriatal dopamine (DA) neurodegeneration. This study aimed to identify disease-modifying treatments for PD. Unsubstituted phenothiazine (PTZ) is a small and uncharged aromatic imine that readily crosses the blood-brain barrier. PTZ lacks significant DA receptor-binding activity and, in the nanomolar range, exhibits protective effects via its potent free radical scavenging and anti-inflammatory activities. Given that DAergic neurons are highly vulnerable to oxidative damage and inflammation, we hypothesized that administration of PTZ might confer neuroprotection in different experimental models of PD. Our findings showed that PTZ rescues rotenone (ROT) toxicity in primary ventral midbrain neuronal cultures by preserving neuronal integrity and reducing protein thiol oxidation. Long-term treatment with PTZ improved animal weight, survival rate, and behavioral deficits in ROT-lesioned rats. PTZ protected DA content and fiber density in the striatum and DA neurons in the SN against the deleterious effects of ROT. Mitochondrial dysfunction, axonal impairment, oxidative insult, and inflammatory response were attenuated with PTZ therapy. Furthermore, we have provided a new insight into the molecular mechanism underlying the neuroprotective effects of PTZ.

Primary Embryonic Rat Cortical Neuronal Culture and Chronic Rotenone Treatment in Microfluidic Culture Devices.

In the study of neurodegenerative diseases, it is imperative to study the cellular and molecular changes associated with pathogenesis in the relevant cell type, central nervous system neurons. The unique compartmentalized morphology and bioenergetic needs of primary neurons present complications for their study in culture. Recent microculture techniques utilizing microfluidic culture devices allows for environmental separation and analysis of neuronal cell bodies and neurites in culture. Here, we present our protocol for culture of primary neurons in microfluidic devices and their chronic treatment with the Parkinson's disease (PD) relevant toxicant rotenone. In addition, we present a method for reuse of devices for culture. This culture methodology presents advantages for evaluating early pathogenic cellular and molecular changes in neurons in a compartment-specific manner.

Sarm1 induction and accompanying inflammatory response mediates age-dependent susceptibility to rotenone-induced neurotoxicity

Aging is a complex biological process and environmental risk factors like pesticide exposure have been implicated in the increased incidence of age-related neurodegenerative diseases like Parkinson’s disease (PD) but the etiology remains unknown. There is also lack of a proper animal model system to study the progressive effect of these environmental toxins on age-associated neurodegeneration. In this study, we established a drosophila model of aging to study the age-dependent vulnerability to the environmental toxin rotenone that has been implicated in sporadic cases of PD. We demonstrate that age plays a determining role in the increased susceptibility to chronic rotenone exposure that is accompanied by severe locomotor deficits, decreased lifespan and loss of dopaminergic (DA) neurons. Chronic low dose exposure to rotenone results in the rapid induction of the neurodegenerative molecule SARM1/dSarm. Further, the age-dependent dSarm induction is accompanied by a heightened inflammatory response (increased expression of Eiger and Relish) that is independent of reactive oxygen species (ROS) generation in the observed rotenone-induced neurotoxicity. dSarm induction and subsequent locomotor deficits is reversed in the presence of the anti-inflammatory molecule resveratrol. Thus, dSarm and heightened inflammatory responses may play a crucial role in age-dependent vulnerability to the pesticide rotenone thus making it an attractive target to help develop cost-effective therapeutic strategies to prevent ongoing dopaminergic neuronal loss as seen in PD.

Improved neuroprotective effect of resveratrol nanoparticles as evinced by abrogation of rotenone-induced behavioral deficits and oxidative and mitochondrial dysfunctions in rat model of Parkinson's disease.

The objective of the present study was to evaluate the protective effect of resveratrol nanoparticles (NRSV) against rotenone-induced neurodegeneration in rats. NRSV were prepared by temperature-controlled antisolvent precipitation method and characterized for its particle size, shape, and dissolution properties. Moreover, NRSV effects compared with the free resveratrol (RSV). Animals were divided into four groups: (I) control, (II) rotenone (2mg/kg s.c.), (III) RSV (40mg/kg, p.o.) + rotenone, and (IV) NRSV (40mg/kg, p.o.) + rotenone. Animals received treatments 30min before rotenone administration for a period of 35days. Behavioral quantifications were done using rota rod test and rearing behavior after 24h of last dose. Animals were euthanized, and mid brains were isolated for the estimation of tricarboxylic acid cycle enzymes, oxidative measures (lipid peroxidation (LPO), glutathione (GSH), and catalase), and complex-I activity. In addition, histopathological studies were also performed. Our results showed that chronic rotenone treatment causes motor deficits, decreased rearing behavior, mitochondrial dysfunction, and oxidative stress. Furthermore, histological analysis demonstrated neuronal degeneration in rotenone-treated rats. An important finding of the present study was NRSV showed comparatively better efficacy than the RSV treatment in attenuating the rotenone-induced Parkinson's like behavioral alterations, biochemical and histological changes, oxidative stress, and mitochondrial dysfunction in rats.

Mitochondrial impairment in microglia amplifies NLRP3 inflammasome proinflammatory signaling in cell culture and animal models of Parkinson\u2019s disease

The NLRP3 inflammasome signaling pathway is a major contributor to the neuroinflammatory process in the central nervous system. Oxidative stress and mitochondrial dysfunction are key pathophysiological processes of many chronic neurodegenerative diseases, including Parkinson’s disease (PD). However, the inter-relationship between mitochondrial defects and neuroinflammation is not well understood. In the present study, we show that impaired mitochondrial function can augment the NLRP3 inflammasome-driven proinflammatory cascade in microglia. Primary mouse microglia treated with the common inflammogen LPS increased NLRP3 and pro-IL-1β expression. Interestingly, exposure of LPS-primed microglial cells to the mitochondrial complex-I inhibitory pesticides rotenone and tebufenpyrad specifically potentiated the NLRP3 induction, ASC speck formation and pro-IL-1β processing to IL-1β in a dose-dependent manner, indicating that mitochondrial impairment heightened the NLRP3 inflammasome-mediated proinflammatory response in microglia. The neurotoxic pesticide-induced NLRP3 inflammasome activation was accompanied by bioenergetic defects and lysosomal dysfunction in microglia. Furthermore, the pesticides enhanced mitochondrial ROS generation in primary microglia, while amelioration of mitochondria-derived ROS by the mitochondria-targeted antioxidant mito-apocynin completely abolished IL-1β release, indicating mitochondrial ROS drives potentiation of the NLRP3 inflammasome in microglia. Exposure to conditioned media obtained from mitochondrial inhibitor-treated, LPS-primed microglial cells, but not unprimed cells, induced dopaminergic neurodegeneration in cultured primary mesencephalic and human dopaminergic neuronal cells (LUHMES). Notably, our in vivo results with chronic rotenone rodent models of PD further support the activation of proinflammatory NLRP3 inflammasome signaling due to mitochondrial dysfunction. Collectively, our results demonstrate that mitochondrial impairment in microglia can amplify NLRP3 inflammasome signaling, which augments the dopaminergic neurodegenerative process.

Combined Oral Supplementation of Fish Oil and Quercetin Enhances Neuroprotection in a Chronic Rotenone Rat Model: Relevance to Parkinson’s Disease

While the neuromodulatory efficacy of n-3 polyunsaturated fatty acids present in fish and fish oil (FO) are well known, some evidence in animal models suggests that chronic consumption of FO supplements may predispose the brain to lipid peroxidation. In view of this, recent approaches envisage the use of dietary antioxidants as adjuncts with FO to obtain a better clinical outcome in neurological disorders. In furtherance of our previous work, in the present study, we examined whether enrichment of FO with quercetin (Q) would enhance the neuroprotective outcome employing a chronic rotenone (ROT) model of neurotoxicity in rats. Growing male rats supplemented either with FO (2 mL/kg bw) or Q (25 mg/kg bw) or FO + Q for 28 days were administered with ROT (0.5 mg/kg bw, 21 days). Monitoring the behavioral phenotype by a battery of tests, terminally, oxidative response in brain regions, mitochondrial dysfunctions and striatal dopamine levels were determined. While both FO and Q offered varying degree of protection, the FO + Q combination offered a higher degree of protection. FO + Q combination significantly attenuated behavioral impairments, restored the ROT-induced oxidative markers, depleted dopamine levels in striatum and reduced mitochondrial dysfunction. These salient findings besides corroborating with our previous data suggest that enrichment of FO with Q indeed offers a higher degree of neuroprotection under chronic exposure to a model neurotoxin such as ROT. Hence, we propose that a combination of FO with known antioxidants such as quercetin is more likely to provide a superior therapeutic advantage in the prevention/treatment of oxidative stress-mediated neurodegenerative conditions such as Parkinson's disease.

Unexpected Improvements of Spatial Learning and Memory Abilities in Chronic Rotenone Intoxicated Mice

The liposoluble insecticide rotenone is commonly used as a mitochondrial complex I inhibitor to replicate Parkinson's disease (PD) pathological features. However, there was no assessment of the spatial learning and memory abilities in chronic rotenone-induced PD models. In the present study, by rotarod test and Thioflavine T staining, we first noted the impairment of motor coordination in rotenone-treated group for 3 months, as well as alpha-synuclein inclusions in the nigral dopaminergic neurons in C57BL/6 mice with intragastrical delivery of rotenone (5 mg/Kg) for 3 months rather than 1 month. We then evaluated spatial learning and memory abilities by Morris water maze task in this model. The results showed escape latency reduced in rotenone-intoxicated mice for 3 months, indicating an improvement of learning ability. However, it was delayed slightly but not significantly in rotenone-intoxicated mice for 1 month. Similarly, we demonstrated that spatial memory ability was enhanced in 3-month-treatment group, but impaired in 1-month-treatment group. There were no proliferating cell nuclear antigen and doublecortin positive cells in the hippocampus by double immunofluorescent staining, indicating the absence of hippocampal neurogenesis in rotenone-intoxicated mice. These results suggest that spatial learning and memory abilities are disturbed in chronic rotenone-intoxicated PD model.

Inhibition of endoplasmic reticulum stress is involved in the neuroprotective effects of candesartan cilexitil in the rotenone rat model of Parkinson's disease

Recent studies indicated that angiotensin II (Ang II) receptor blockers could reduce neurotoxins-induced dopaminergic (DA) cell death, but the underlying mechanisms are still unclear. Given that endoplasmic reticulum (ER) stress plays a major role in rotenone-induced neuronal apoptosis, we investigated whether candesartan cilexetil, a selective and high-affinity Ang II receptor antagonist, could protect the DA neuron via reducing ER stress in a chronic rotenone rat model for Parkinson's disease (PD). Our data showed that candesartan cilexetil could ameliorate the descent latency in catalepsy tests, and decrease rotenone-induced DA neuron apoptosis. Moreover, candesartan cilexetil has been found to play a protective role via down-regulating the expression of activating transcription factor 4 (ATF4), the CCAAT-enhancer-binding protein (C/EBP) homologous protein (CHOP), and p53 upregulated modulator of apoptosis (Puma). Thus, our experiments strongly suggest that administration of candesartan cilexetil protects DA neuron involving blocking ER stress, possibly via inhibiting activation of the ATF4–CHOP–Puma pathway, which could provide new insight into clinical therapeutics for PD.

Chronic oxidative stress modulates TRPC3 and TRPM2 channel expression and function in rat primary cortical neurons: Relevance to the pathophysiology of bipolar disorder

Recent findings implicate the calcium-permeable transient receptor potential (TRP) melastatin subtype 2 (TRPM2) and canonical subtype 3 (TRPC3) channels in the pathogenesis of bipolar disorder (BD). As both channels are involved in calcium and oxidative stress signaling, thought to be disrupted in BD, we sought to determine the effects of elevated oxidative stress on their expression and function. Primary rat cortical neurons and astrocytes were treated with oxidative stressors for 1 (acute) and 4 days (chronic). Expression of TRPC3 and TRPM2 were determined by immunoblotting and real-time PCR. Channel functionality was assessed using a TRPC3 activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and live cell, ratiometric fluorometry with the calcium sensitive dye, Fura-2. Neurons treated with rotenone (15–30nM) for 4 days but not 24h showed significant dose-dependent decreases in TRPC3 mRNA (31%, p<0.001) and protein levels (60%, p<0.001). Similar dose-dependent attenuation of TRPC3-mediated calcium fluxes was demonstrated upon chronic rotenone exposure relative to vehicle controls. In contrast, TRPM2 mRNA but not protein levels increased (47%, p=0.017) after acute and chronic rotenone treatment. Chronic exposure of neurons to paraquat (1–2µM), an alternate oxidative stressor, similarly decreased TRPC3 expression (mRNA: 41%; protein: 61%). Unlike neurons, rotenone treatment incurred no changes in astrocyte TRPC3 levels. These findings demonstrate that TRPC3 and TRPM2 channel expression and/or function is sensitive to the redox status of rat primary neurons and that these changes are time dependent. This provides a critical mechanistic link between altered oxidative stress markers, dysfunction of these TRP channels and calcium dyshomeostasis in BD.

Neurochemical changes in animal models of Parkinson’s disease

Parkinson’s disease (PD) is a neurodegenerative syndrome associated with selective loss of dopaminergic neurons in the striatum. It is believed that both mitochondrial dysfunction and oxidative stress (OS) play important role in the pathogenesis of PD. Development of an effective causal therapy should be focused on preventing or at least retarding the neurodegenerative process underlying the disease. In chronic in fusion PD model studied the effect of rotenone on the content, and release of striatal neurotransmitters of rat upon OS by H2O 2 perfusion. The effects of anti-Parkinsonian drugs were examined in vitro rotenone PD, OS slices model. The role of P2X7 receptor (P2X7r) formed by 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) or in vitro rotenone treatment using C57/BI6 and P2X7r knockout mice. Neurochemical response was determined using microvolume perfusion method, HPLC separation of dansylated samples. Dopamine o-quinone (DAQ) oxidized metabolite of DA was detected electrochemically at -100 mV potential in reduction working mode. The dopamine content in the rat striatum is decreased in response to chronic intravenous rotenone infusion. However, surviving dopaminergic neurons take up and release only a slightly lower amount of DA in response to electrical stimulation. Striatal dopaminergic neurons showed increased susceptibility to OS, responding with enhanced release of DA and with formation of toxic metabolite DAQ. The loss of DA and ATP and formation of DAQ induced by OS confirmed the impairment of striatum upon in vitro rotenone. Formation of DAQ at the end effect of OS confirmed the impairment of dopaminergic function by rotenone. However, when applied rotenone with l-DOPA treatment the endogenous DAQ was detected in perfusion samples. However, when applied rotenone with l-DOPA treatment the endogenous DAQ was detected in the micro-volume perfusion samples. Conversely, when rasagiline and trolox were in use these DA-quinones metabolites were not detectable. Rotenone treatment elicited a similar reduction in ATP content in the substantia nigra of both genotypes, whereas reduction of ATP was more pronounced after rotenone treatment in striatal slices of P2X7 deficient mice. Although the level of the endocannabinoid, 2-AG, was elevated by rotenone in the striatum of wild-type mice, an effect that was absent in mice deficient in P2X7 receptors. Genetic deletion of P2X7 receptors did not change depletion of striatal endogenous DA content after in vivo MPTP or in vitro rotenone treatment. Although the endogenous amino acids content and the level of the endocannabinoid, AEA was elevated by MPTP in the striatum of mice, but 2-AG remained unchanged in mice deficient in P2X7 receptors. In conclusion, oxidative stress induced, pathological DA release and the formation of toxic metabolites of DA are distinctly modified by the tested antiparkinsonian drugs and P2X7 receptor deficiency does not promote the survival of dopaminergic neurons.

Anti-inflammatory properties rather than anti-oxidant capability is the major mechanism of neuroprotection by sodium salicylate in a chronic rotenone model of Parkinson’s disease

Parkinson’s disease (PD) is the second most common neurodegenerative disorder manifesting in motor, cognitive and behavioral anomalies. Loss of dopaminergic neurons in the substantia nigra region of the brain is the hallmark feature of PD, which is attributed to oxidative and inflammatory stress besides other diverse factors and hence drugs targeting these pathways hold promise as neuro-therapeutics.The anti-oxidative as well as anti-inflammatory properties of sodium salicylate (SS), suggest its neuroprotective potentials in PD. Since PD is a progressive neurodegenerative disorder, the mechanistic basis for utilizing SS as a neuroprotectant in PD could be better understood in the chronic models. The present study utilizes a rotenone-based model of PD to evaluate the neuro-modulatory efficacy of SS. Subcutaneous injection of rotenone (2mg/kg body weight) was given to male SD rats every day, for a period of 5weeks, which developed all the essential features of PD in these animals. Simultaneously, another group was injected SS intraperitoneally at the dose of 100mg/kg body weight, in addition to the rotenone. In the animals receiving rotenone+SS, significant improvement was observed in the various characteristic hallmarks of PD such as dopamine and tyrosine hydroxylase levels as well as the motor dysfunction symptoms. It attenuated the reactive oxygen species levels significantly but failed to reduce the levels of protein carbonylation and lipid peroxidation. However, SS effectively abridged the levels of inflammatory mediators like cyclooxygenase-2 (COX-2), nuclear factor kappa B and inducible nitric oxide synthase. Correspondingly, a significant decrease in the levels of pro-inflammatory cytokines interleukin-6, interleukin-1β and tumor necrosis factor-α was also observed following SS co-treatment. Thus, neuroprotective efficacy of SS in this chronic model of PD can be largely attributed to its anti-inflammatory effects rather than its free radical-scavenging properties.

Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects

Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP(+)-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP(+)-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP(+)-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Mitochondrial Dysfunction and Oxidative Stress Promote Apoptotic Cell Death in the Striatum via Cytochrome c/ Caspase-3 Signaling Cascade Following Chronic Rotenone Intoxication in Rats

Parkinson’s disease (PD) is a progressive neurological disorder marked by nigrostriatal dopaminergic degeneration. Evidence suggests that mitochondrial dysfunction may be linked to PD through a variety of different pathways, including free-radical generation and dysfunction of the mitochondrial Complex I activity. In Lewis rats, chronic systemic administration of a specific mitochondrial Complex I inhibitor, rotenone (3 mg/kg/day) produced parkinsonism-like symptoms. Increased oxidized proteins and peroxynitrite, and mitochondrial or cytosol translocation of Bim, Bax or cytochrome c in the striatum was observed after 2–4 weeks of rotenone infusion. After 28 days of systemic rotenone exposure, imunohistochemical staining for tyrosine hydroxylase indicated nigrostriatal dopaminergic neuronal cell degeneration. Characteristic histochemical (TUNEL or activated caspase-3 staining) or ultrastructural (electron microscopy) features of apoptotic cell death were present in the striatal neuronal cell after chronic rotenone intoxication. We conclude that chronic rotenone intoxication may enhance oxidative and nitrosative stress that induces mitochondrial dysfunction and ultrastructural damage, resulting in translocation of Bim and Bax from cytosol to mitochondria that contributes to apoptotic cell death in the striatum via cytochrome c/caspase-3 signaling cascade.

Rotenone-induced parkinsonism elicits behavioral impairments and differential expression of parkin, heat shock proteins and caspases in the rat

Rotenone is a pesticide that inhibits mitochondrial complex I activity, thus creating an environment of oxidative stress in the cell. Many studies have employed rotenone to generate an experimental animal model of Parkinson’s disease (PD) that mimics and elicits PD-like symptoms, such as motor and cognitive decline. Cytoprotective proteins including parkin and heat shock proteins (HSPs) play major roles in slowing PD progression. Moreover, evidence suggests that mitochondrial dysfunction and oxidative stress-dependent apoptotic pathways contribute to dopaminergic neuron degeneration in PD. Here, rats were chronically exposed to rotenone to confirm that it causes a debilitating phenotype and various behavioral defects. We also performed histopathological examinations of nigrostriatal, cortical and cerebellar regions of rotenone-treated brain to elucidate a plausible neurodegenerative mechanism. The results of silver, tyrosine hydroxylase (TH), parkin, ubiquitin and caspase staining of brain tissue sections further validated our findings. The stress response is known to trigger HSP in response to pharmacological insult. These protective proteins help maintain cellular homeostasis and may be capable of rescuing cells from death. Therefore, we assessed the levels of different HSPs in the rotenone-treated animals. Collectively, our studies indicated the following findings in the striatum and substantia nigra following chronic rotenone exposure in an experimental PD model: (i) behavioral deficit that correlated with histopathological changes and down regulation of TH signaling, (ii) decreased levels of the cytoprotective proteins parkin, DJ1 and Hsp70 and robust expression of mitochondrial chaperone Hsp60 according to Western blot, (iii) increased immunoreactivity for caspase 9, caspase 3 and ubiquitin and decreased parkin immunoreactivity.