MPTP-induced Toxicity Research Articles

Artemisinin protects dopaminergic neurons against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity in a mouse model of Parkinson’s disease

Artemisinin is an antimalarial drug that has been used for almost half a century. However, the anti-Parkinson's disease (PD) effects of artemisinin with respect to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced oxidative stress have not yet been investigated while focusing on NF-E2–related factor 2 (Nrf2) signaling. Thus, we sought to assess the behavioral and oxidative mechanistic effects of artemisinin on MPTP-induced toxicity via the Nrf2 signaling pathway. We explored this through immunohistochemical assays, ELISA, in differentiated PC12 cells treated with siRNA, and with a PD mouse model. Artemisinin increased Nrf2 DNA-binding activity and HO-1 and NQO1 expression. Artemisinin treatment protected cells against MPP+ -induced neuronal death signaling, including NADH dehydrogenase activity, reactive oxygen species, mitochondrial membrane potential, and cleaved caspase-3. Moreover, it protected cells against MPTP-induced behavioral impairments and significantly reduced dopaminergic neuronal loss. Additionally, Nrf2 pre-inhibition using ML385 neutralized the inhibitory effects of artemisinin on dopaminergic neuronal damage and behavioral impairments induced by MPTP. Our results suggest that artemisinin inhibits MPTP-induced behavioral and neurotoxic effects in mice. This provides a foundation for further research to evaluate artemisinin as a potential therapeutic agent for PD.

Biological interactions and attenuation of MPTP-induced toxicity in Drosophila melanogaster by Trans-astaxanthin

Trans-astaxanthin (TA) is a carotenoid with amphipathic chemical structure found in yeast, and aquatic organisms. It is known to possess both antioxidative and anti-inflammatory properties. This study was carried out to investigate the ameliorative action of TA on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity in Drosophila melanogaster (Fruit fly). The flies were orally treated with TA (2.5 mg/10 g diet) and/or MPTP (500 µM) for 5 days. Thereafter, we evaluated selected biomarkers of locomotor deficits (acetylcholinesterase (AChE) and negative geotaxis), oxidative stress (hydrogen peroxide (H2O2), protein carbonyls (PC)), antioxidants (total thiols (T-SH), non-protein thiols, glutathione-S-transferase (GST) and catalase), and inflammation (nitric oxide (nitrite/nitrate) in the flies. Furthermore, we investigated molecular docking analysis of TA against Kelch-like ECH-associated protein 1 (Keap1)) of Homo sapiens and D. melanogaster. The results indicated that TA increased MPTP-induced decreased activities of AChE, GST, and catalase, as well as levels of non-protein thiols and T-SH compared with MPTP-treated flies (p < 0.05). Furthermore, TA attenuated inflammation, and improved locomotor deficit in the flies. The molecular docking data showed that TA had docking scores for binding both the Human and Drosophila Keap1, nearly closer to or higher than the standard inhibitor. The attenuating effects of TA against MPTP-induced toxicity could arise from its antioxidative and anti-inflammatory properties as well as its chemical structure.

Microglial cGAS drives neuroinflammation in the MPTP mouse models of Parkinson's disease.

Neuroinflammation has been widely accepted as a cause of the degenerative process. Increasing interest has been devoted to developing intervening therapeutics for preventing neuroinflammation in Parkinson's disease (PD). It is well known that virus infections, including DNA viruses, are associated with an increased risk of PD. In addition, damaged or dying dopaminergic neurons can release dsDNA during PD progression. However, the role of cGAS, a cytosolic dsDNA sensor, in PD progression remains unclear. Adult male wild-type mice and age-matched male cGAS knockout (cGas-/- ) mice were treated with MPTP to induce neurotoxic PD model, and then behavioral tests, immunohistochemistry, and ELISA were conducted to compare disease phenotype. Chimeric mice were reconstituted to explore the effects of cGAS deficiency in peripheral immune cells or CNS resident cells on MPTP-induced toxicity. RNA sequencing was used to dissect the mechanistic role of microglial cGAS in MPTP-induced toxicity. cGAS inhibitor administration was conducted to study whether GAS may serve as a therapeutic target. We observed that the cGAS-STING pathway was activated during neuroinflammation in MPTP mouse models of PD. cGAS deficiency in microglia, but not peripheral immune cells, controlled neuroinflammation and neurotoxicity induced by MPTP. Mechanistically, microglial cGAS ablation alleviated the neuronal dysfunction and inflammatory response in astrocytes and microglia by inhibiting antiviral inflammatory signaling. Additionally, the administration of cGAS inhibitors conferred the mice neuroprotection during MPTP exposure. Collectively, these findings demonstrate microglial cGAS promote neuroinflammation and neurodegeneration during the progression of MPTP-induced PD mouse models and suggest cGAS may serve as a therapeutic target for PD patients. Although we demonstrated that cGAS promotes the progression of MPTP-induced PD, this study has limitations. We identified that cGAS in microglia accelerate disease progression of PD by using bone marrow chimeric experiments and analyzing cGAS expression in CNS cells, but evidence would be more straightforward if conditional knockout mice were used. This study contributed to the knowledge of the role of the cGAS pathway in PD pathogenesis; nevertheless, trying more PD animal models in the future will help us to understand the disease progression deeper and explore possible treatments.

A Newly Identified Monoterpenoid-Based Small Molecule Able to Support the Survival of Primary Cultured Dopamine Neurons and Alleviate MPTP-Induced Toxicity In Vivo

Parkinson's disease (PD) is the most common age-related movement disorder characterized by the progressive loss of nigrostriatal dopaminergic neurons. To date, PD treatment strategies are mostly based on dopamine replacement medicines, which can alleviate motor symptoms but do not slow down the progression of neurodegeneration. Thus, there is a need for disease-modifying PD therapies. The aim of this work was to evaluate the neuroprotective effects of the novel compound PA96 on dopamine neurons in vivo and in vitro, assess its ability to alleviate motor deficits in MPTP- and haloperidol-based PD models, as well as PK profile and BBB penetration. PA96 was synthesized from (1R,2R,6S)-3-methyl-6-(prop-1-en-2-yl) cyclohex-3-ene-1,2-diol (Prottremin) using the original three-step stereoselective procedure. We found that PA96: (1) supported the survival of cultured näive dopamine neurons; (2) supported the survival of MPP+-challenged dopamine neurons in vitro and in vivo; (3) had chemically appropriate properties (synthesis, solubility, etc.); (4) alleviated motor deficits in MPTP- and haloperidol-based models of PD; (5) penetrated the blood-brain barrier in vivo; and (6) was eliminated from the bloodstream relative rapidly. In conclusion, the present article demonstrates the identification of PA96 as a lead compound for the future development of this compound into a clinically used drug.

Additive cell protective and oxidative stress reducing effects of combined treatment with cromolyn sodium and masitinib on MPTP-induced toxicity in SH-SY5Y neuroblastoma cells

The suppression of oxidative-stress induced neurotoxicity by antioxidants serves as a potential preventive strategy for neurodegenerative diseases. In this study, we aimed to investigate the cell protective and antioxidant effects of masitinib and cromolyn sodium against toxin-induced neurodegeneration.First, human neuroblastoma SH-SY5Y cells were differentiated into neuron-like (d)-SH-SY5Y cells. The differentiated cells were confirmed by immuno-staining with anti-PGP9.5 antibody, a neuronal marker. Cell culture groups were formed, and a neurotoxin, 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP) was applied on cells followed by masitinib and/or cromolyn sodium treatments. Survival rate of cells were detected by MTT assay. Anti-inflammatory Transforming Growth Factor-β1 (TGF-β1) and nitric oxide (NO) levels and total oxidant and antioxidant capacities (TOC and TAC) in cell conditioned media (CM) were measured. Morphological analysis and apoptotic nuclear assessment of cells were also noted.When (d)-SH-SY5Y cells were exposed to neurotoxin, cell viability rates of these cells were found to be decreased in a concentration-dependent manner. CM of toxin applied group displayed higher levels of TOC/TAC ratios and NO levels compared to control (p < 0.01). Both masitinib and cromolyn sodium protected cells from toxin-induced cell death as revealed by ameliorated rates of viability, reversed toxin-induced elevation of TOC/TAC ratios, and decreased NO levels in their CM (p < 0.01). Combined treatment significantly reduced TOC/TAC ratios and NO levels more effectively compared to mono-treatments. Both drugs also increased TGF-β1 levels significantly in cell CM. When these agents were tested for therapeutic effects against toxin-induced cell degeneration, better viability results were obtained by both masitinib and cromolyn sodium treatment, with significantly better amelioration provided by combined application of these drugs (p < 0.01).This study demonstrated new findings that combined treatment with cromolyn sodium, an FDA-approved drug of asthma, and masitinib, an orally administered drug with a low toxicity, exert neuroprotective and additive therapeutic effects. We propose that combination therapy of masitinib and cromolyn sodium may represent an innovative treatment in neurodegenerative diseases. Combination therapy may be more advantageous that it enables combined application of lower doses of both drugs, providing less side effects.

Aged xCT-Deficient Mice Are Less Susceptible for Lactacystin-, but Not 1-Methyl-4-Phenyl-1,2,3,6- Tetrahydropyridine-, Induced Degeneration of the Nigrostriatal Pathway.

The astrocytic cystine/glutamate antiporter system xc– (with xCT as the specific subunit) imports cystine in exchange for glutamate and has been shown to interact with multiple pathways in the brain that are dysregulated in age-related neurological disorders, including glutamate homeostasis, redox balance, and neuroinflammation. In the current study, we investigated the effect of genetic xCT deletion on lactacystin (LAC)- and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced degeneration of the nigrostriatal pathway, as models for Parkinson’s disease (PD). Dopaminergic neurons of adult xCT knock-out mice (xCT–/–) demonstrated an equal susceptibility to intranigral injection of the proteasome inhibitor LAC, as their wild-type (xCT+/+) littermates. Contrary to adult mice, aged xCT–/– mice showed a significant decrease in LAC-induced degeneration of nigral dopaminergic neurons, depletion of striatal dopamine (DA) and neuroinflammatory reaction, compared to age-matched xCT+/+ littermates. Given this age-related protection, we further investigated the sensitivity of aged xCT–/– mice to chronic and progressive MPTP treatment. However, in accordance with our previous observations in adult mice (Bentea et al., 2015a), xCT deletion did not confer protection against MPTP-induced nigrostriatal degeneration in aged mice. We observed an increased loss of nigral dopaminergic neurons, but equal striatal DA denervation, in MPTP-treated aged xCT–/– mice when compared to age-matched xCT+/+ littermates. To conclude, we reveal age-related protection against proteasome inhibition-induced nigrostriatal degeneration in xCT–/– mice, while xCT deletion failed to protect nigral dopaminergic neurons of aged mice against MPTP-induced toxicity. Our findings thereby provide new insights into the role of system xc– in mechanisms of dopaminergic cell loss and its interaction with aging.

Caffeine, a natural methylxanthine nutraceutical, exerts dopaminergic neuroprotection

Parkinson's disease (PD) is a progressive neurodegenerative disorder that affects more than 10 million people worldwide. Oxidative stress and mitochondrial dysfunction play a significant role in altering the homeostasis of energy production and free radical generation. Current PD therapies are focused on reducing the cardinal symptoms rather than preventing disease progression in the patients. Adenosine A2A receptor (A2A R) antagonist (Istradephylline) combined with levodopa shows a promising therapy for PD. In animal studies, caffeine administration showed to improve motor functions and neuroprotective effect in the neurons. Caffeine is probably the most extensively used psychoactive substance. In this current study, we investigated the neuroprotective effect of caffeine against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurodegeneration. Here, we demonstrate that caffeine improves behavioral and neurotransmitter recovery against MPTP-induced toxicity. Caffeine restores endogenous antioxidant levels and suppresses neuroinflammation. Our finding suggests that the blockage of A2AR is a promising disease-modifying therapy for PD. Target engagement strategies could be more beneficial in preventing disease progression rather than symptomatic reliefs in PD patients.

The insect molting hormone 20-hydroxyecdysone protects dopaminergic neurons against MPTP-induced neurotoxicity in a mouse model of Parkinson's disease

20-hydroxyecdysone (20E), a steroidal prohormone, is secreted from the prothoracic glands. While 20E has been shown to have neuroprotective effects in Parkinson's disease (PD) models in vitro, its effects have not yet been examined in vivo. We sought to assess the behavioral and mechanistic effects of 20E on MPTP-induced toxicity in mice. To this end, we used behavioral tests, stereological analyses of dopaminergic neurons by tyrosine hydroxylase immunohistochemistry, and assessments of apoptotic mechanisms, focusing on Nrf2 signaling through Western blotting and ELISA assays. A 20E treatment protected against MPTP-induced motor incoordination, postural imbalance, and bradykinesia, and significantly reduced dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc) and the striatum (ST). It also attenuated dopamine deficiency in the ST, modulated levels of antioxidative enzymes superoxide dismutase, catalase, and glutathione in the SNpc, increased the Bcl-2/Bax ratio, and inhibited cytosolic cytochrome c release and caspase-9, -7, and -3 activity in the SNpc. These results indicated that 20E inhibited the apoptotic cascade. Furthermore, the attenuation of MPTP neurotoxicity was associated with inhibited cleaved-caspase signaling pathways, along with upregulated Nrf2 pathways in the SNpc, suggesting that 20E mitigates MPTP-induced neurotoxicity via mitochondria-mediated apoptosis by modulating anti-oxidative activities. Our results suggest that 20E can inhibit MPTP-induced behavioral and neurotoxic effects in mice. This lays the foundation for further research on 20E as a potential target for therapeutic use.

LY354740 Reduces Extracellular Glutamate Concentration, Inhibits Phosphorylation of Fyn/NMDARs, and Expression of PLK2/pS129 α-Synuclein in Mice Treated With Acute or Sub-Acute MPTP.

Glutamate overactivity in basal ganglia critically contributes to the exacerbation of dopaminergic neuron degeneration in Parkinson’s disease (PD). Activation of group II metabotropic glutamate receptors (mGlu2/3 receptors), which can decrease excitatory glutamate neurotransmission, provides an opportunity to slow down the degeneration of the dopaminergic system. However, the roles of mGlu2/3 receptors in relation to PD pathology were partially recognized. By using mGlu2/3 receptors agonist (LY354740) and mGlu2/3 receptors antagonist (LY341495) in mice challenged with different cumulative doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we demonstrated that systemic injection of LY354740 reduced the level of extracellular glutamate and the extent of nigro-striatal degeneration in both acute and sub-acute MPTP mice, while LY341495 amplified the lesions in sub-acute MPTP mice only. LY354740 treatment improved behavioral dysfunctions mainly in acute MPTP mice and LY341495 treatment seemed to aggravate motor deficits in sub-acute MPTP mice. In addition, ligands of mGlu2/3 receptors also influenced the total amount of glutamate and dopamine in brain tissue. Interestingly, compared with normal mice, MPTP-treated mice abnormally up-regulated the expression of polo-like kinase 2 (PLK2)/pS129 α-synuclein and phosphorylation of Fyn/N-methyl-D-aspartate receptor subunit 2A/2B (GluN2A/2B). Both acute and sub-acute MPTP mice treated with LY354740 dose-dependently reduced all the above abnormal expression. Compared with MPTP mice treated with vehicle, mice pretreated with LY341495 exhibited much higher expression of p-Fyn Tyr416/p-GluN2B Tyr1472 and PLK2/pS129 α-synuclein in sub-acute MPTP mice models. Thus, our current data indicated that mGlu2/3 receptors ligands could influence MPTP-induced toxicity, which supported a role for mGlu2/3 receptors in PD pathogenesis.

Mercury sulfide-containing Hua-Feng-Dan and 70W (Rannasangpei) protect against LPS plus MPTP-induced neurotoxicity and disturbance of gut microbiota in mice

Ethnopharmacological relevanceMercury sulfides (HgS) are frequently included in Ayurveda, Tibetan and Chinese medicines to assist the presumed therapeutic effects, but the ethnopharmacology remains elusive. The present study examined the protective effects of α-HgS-containing Hua-Feng-Dan and β-HgS-containing 70 Wei-Zhen-Zhu-Wan (70W, Rannasangpei) against Parkinson's disease mice induced by lipopolysaccharide (LPS) plus 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). MethodA single injection of LPS (5 mg/kg ip) was given to adult male C57BL/6 mice, and 150 days later, the low dose of MPTP (15 mg/kg, ip, for 4 days) was given to produce the “two-hit” Parkinson's disease model. Together with MPTP treatment, mice were fed with clinically-relevant doses of Hua-Feng-Dan (0.6 g/kg) and 70W (0.2 g/kg) for 35 days. Rotarod test was performed to examine muscle coordination capability. At the end of the experiment, brain was transcardially perfused with paraformaldehyde, the substantia nigra was sectioned for microglia (Iba1 staining) and dopaminergic neuron (THir staining) determination. Colon bacterial DNA was extracted and subjected to qPCR analysis with 16S rRNA probes. ResultsThe low-grade, chronic neuroinflammation produced by LPS aggravated MPTP neurotoxicity, as evidenced by decreased motor activity, intensified microglia activation and loss of dopaminergic neurons. Both Hua-Feng-Dan and 70W increased rotarod activity and ameliorated the pathological lesions in the brain. In gut microbiomes examined, LPS plus MPTP increased Verrucomicrobiaceae, Methanobacteriaceae, Pronicromonosporaceae, and Clostridaceae species were attenuated by Hua-Feng-Dan and 70W. Conclusionsα-HgS-containing Hua-Feng-Dan and β-HgS-containing 70W at clinical doses protected against chronic LPS plus MPTP-induced toxicity to the brain and gut, suggesting HgS-containing traditional medicines could target gut microbiota as a mechanism of their therapeutic effects.

Batryticatus Bombyx Protects Dopaminergic Neurons Against MPTP-Induced Neurotoxicity by Inhibiting Oxidative Damage.

Oxidative stress plays an important role in the degeneration of dopaminergic neurons in Parkinson’s disease (PD). Altered redox homeostasis in neurons interferes with several biological processes, ultimately leading to neuronal death. Oxidative damage has been identified as one of the principal mechanisms underlying the progression of PD. Several studies highlight the key role of superoxide radicals in inducing neuronal toxicity. Batryticatus Bombyx (BB), the dried larva of Bombyx mori L. infected by Beauveria bassiana (Bals.) Vuill., has been used in traditional medicine for its various pharmacological effects. In the present study, BB showed a beneficial effect on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity by directly targeting dopaminergic neurons. Treatment with BB improved behavioral impairments, protected dopaminergic neurons, and maintained dopamine levels in PD mouse models. Here, we investigated the protective effects of BB on MPTP-induced PD in mice and explored the underlying mechanisms of action, focusing on oxidative signaling. In MPTP-induced PD, BB promoted recovery from impaired movement, prevented dopamine depletion, and protected against dopaminergic neuronal degradation in the substantia nigra pars compacta (SNpc) or the striatum (ST). Moreover, BB upregulated mediators of antioxidative response such as superoxidase dismutase (SOD), catalase (CAT), glutathione (GSH), Heme oxygenase 1 (HO-1), and NAD(P)H (nicotinamide adenine dinucleotide phosphate) dehydrogenase (NQO1). Thus, treatment with BB reduced the oxidative stress, improved behavioral impairments, and protected against dopamine depletion in MPTP-induced toxicity.

Luteolin-7-O-glucoside protects dopaminergic neurons by activating estrogen-receptor-mediated signaling pathway in MPTP-induced mice

BackgroundParkinson's disease (PD) is a neurodegenerative disorder that is characterized by the degeneration of dopaminergic neurons in substantia nigra (SN). Accumulating evidences implicate the beneficial role of estrogen in the therapy of PD. MethodsIn the present study, the protective function of luteolin-7-O-glucoside (LUT-7G), a natural flavonoid, was investigated in 1-methyl-4-phenylpyridinium (MPP+) treated SH-SY5Y cells and 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) induced mice. ResultsPre-treatment of LUT-7G increased the viability and reduced the apoptosis of SH-SY5Y cells treated by MPP+. At molecular level, the Bcl-2/Bax ratio was increased, while the expression of cleaved caspase 3 was markedly lessened. Moreover, LUT-7G increased the expression of estrogen receptor (ER), ERα and ERβ, and enhanced the activation of ERK1/2/STAT3/c-Fos that could be abolished by ER antagonists. Furthermore, in vivo experiment indicated that pre-treatment of LUT-7G improved the bradykinesia, and enhanced the muscle strength as well as the balancing capacity of mice treated with MPTP. And LUT-7G prevented the injury of TH positive cells in substantia nigra and increased TH positive nerve fibers in striatum. In addition, pre-treatment of LUT-7G also significantly diminished the MPTP-induced gliosis in substantia nigra. ConclusionsLUT-7G effectively protected dopaminergic neurons against MPP+ or MPTP-induced toxicity, probably by activating the ER-mediated signaling pathway. Our findings explore the therapeutic potential of LUT-7G for PD therapy.

Lipostatic Mechanisms Preserving Cerebellar Lipids in MPTP-Treated Mice: Focus on Membrane Microdomains and Lipid-Related Gene Expression.

The cerebellum is an essential component in the control of motor patterns. Despite dramatic alteration of basal ganglia morpho-functionality in Parkinson’s disease (PD), cerebellar function appears to be unaffected by the disease. Only recently this brain structure has been proposed to play compensatory roles in PD-induced motor dysfunction, particularly during the initial asymptomatic stages of PD. In PD subjects and animal models of PD, such as MPTP-treated mice, brain structures other than basal ganglia are also affected by the disease, including cortical areas not involved in motor control. Thus, it is noteworthy that the cerebellum remains unaffected. In the present study, we have analyzed the lipid composition of membrane microdomains [lipid rafts (LR) and non-raft domains] and assessed the expression levels of genes encoding enzymes synthesizing membrane-related lipids. The outcomes revealed that membrane domain lipids in cerebellum are highly preserved both in control and MPTP-treated mice as compared to control animals. Likewise, only small, mostly not significant, changes were observed in the expression of lipid-related genes in the cerebellum. Indeed, most changes were related to aging rather than to the exposure to the neurotoxin. Conversely, in the same animals, lipid composition, and gene expression were dramatically altered in the occipital cortex (OC), a brain area unrelated to the control of motor function. PCR and immunohistochemical analyses of both brain areas revealed that dopamine transporter (DAT) mRNA and protein were expressed in OC but not in the cerebellum. As MPTP neurotoxicity requires the expression of DAT to access intracellular compartments, we hypothesized that the absence of DAT in cerebellum hampers MPTP-induced toxicity. We conclude that cerebellum is endowed with efficient mechanisms to preserve nerve cell lipid homeostasis, which greatly maintain the stability of membrane microdomains involved in synaptic transmission, signal transduction, and intercellular communication, which together may participate in the compensatory role of the cerebellum in PD symptomatology.

Thymoquinone prevents neurodegeneration against MPTP in vivo and modulates α-synuclein aggregation in vitro

Parkinson's disease (PD) is a common neurodegenerative disease characterized by progressive dopaminergic neurodegeneration with a concomitant increase in oxidative stress and neuroinflammation in the substantia nigra pars compacta (SNc). Recent studies have focused on targeting neuroinflammation and oxidative stress to effectively treat PD. The present study evaluated the neuroprotective effect of thymoquinone (TQ) against 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP)-induced oxidative stress and neuroinflammation in a PD mouse model. TQ (10 mg/kg body weight [b. wt.]) was administered for 1 week prior to MPTP (25 mg/kg b. wt.). MPTP administration caused oxidative stress as evidenced by decreased activities of superoxide dismutase and catalase, a depletion of reduced glutathione, and a concomitant rise in malondialdehyde. It also significantly increased pro-inflammatory cytokines and elevated inflammatory mediators such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the striatum. Immunohistochemical analysis revealed dopamine neuron loss in the SNc and decreased dopamine transporters in the striatum following MPTP administration; however, these were rescued by TQ treatment. TQ treatment further restored antioxidant enzymes, prevented glutathione depletion, inhibited lipid peroxidation, and attenuated pro-inflammatory cytokines. TQ also decreased the raised levels of inflammatory mediators, such as COX-2 and iNOS. Therefore, TQ is thought to protect against MPTP-induced PD and the observed neuroprotective effects are attributed to its potent antioxidant and anti-inflammatory properties. Moreover, the in vitro analysis found that TQ significantly inhibited α-synuclein aggregation and prevented cell death induced by pre-formed fibrils. Thus, TQ not only scavenges the MPTP-induced toxicity but also prevents α-synuclein-fibril formation and its associated toxicity.

Rosmarinic acid protects against MPTP-induced toxicity and inhibits iron-induced α-synuclein aggregation

Rosmarinic acid (RA) is a naturally occurring polyphenolic compound. In this study, we demonstrated that RA could protect against the degeneration of the nigrostriatal dopaminergic system in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of Parkinson's disease (PD). In addition, RA could inhibit MPTP-induced decrease of superoxide dismutase (SOD) and tyrosine hydroxylase (TH) and increase in nigral iron content. Further studies elucidated the effects of RA on iron-induced neurotoxicity and the possible underlying mechanisms in the SK-N-SH cells. Results showed that iron could induce a decrease in the mitochondrial transmembrane potential and result in α-synuclein aggregation in the SK-N-SH cells, which could be restored by RA pretreatment. Further results showed RA pretreatment could inhibit iron-induced α-synuclein aggregation by up-regulating hemeoxygenase-1 (HO-1). In addition, iron could increase the mRNA levels of α-synuclein via iron responsive element/iron regulatory protein (IRE/IRP) system. RA pretreatment could decrease the mRNA levels of α-synuclein by decreasing the protein levels of IRP1. These results indicated that RA protected against iron-induced α-synuclein aggregation by up-regulating HO-1 and inhibiting α-synuclein expression.

Neuroprotective Role of Atractylenolide-I in an In Vitro and In Vivo Model of Parkinson's Disease.

Parkinson’s disease (PD) is an age-related neurological disorder characterized by a loss of dopaminergic neurons within the midbrain. Neuroinflammation has been nominated as one of the key pathogenic features of PD. Recently, the inadequate pharmacotherapy and adverse effects of conventional drugs have spurred the development of unconventional medications in the treatment of PD. The purpose of this study is to investigate the anti-neuroinflammatory mechanisms of Atractylenolide-I (ATR-I) in in vivo and in vitro models of PD. Nitrite assay was measured via Griess reaction in lipopolysaccharide (LPS) stimulated BV-2 cells. mRNA and protein levels were determined by a reverse transcription-polymerase chain reaction (RT-PCR) and immunoblot analysis, respectively. Further, flow cytometry, immunocytochemistry, and immunohistochemistry were employed in BV-2 cells and MPTP-intoxicated C57BL6/J mice. Pre-treatment with ATR-I attenuated the inflammatory response in BV-2 cells by abating the nuclear translocation of nuclear factor-κB (NF-κB) and by inducing heme oxygenase-1 (HO-1). The intraperitoneal administration of ATR-I reversed MPTP-induced behavioral deficits, decreased microglial activation, and conferred protection to dopaminergic neurons in the mouse model of PD. Our experimental reports establish the involvement of multiple benevolent molecular events by ATR-I in MPTP-induced toxicity, which may aid in the development of ATR-I as a new therapeutic agent for the treatment of PD.

Neuroprotection by freshwater clam extract against the neurotoxin MPTP in C57BL/6 mice

Freshwater clams are a popular health food in Asia and are traditionally used to prevent hepatic inflammation. Freshwater clam extract (FCE) inhibits inflammatory responses in activated macrophages by reducing the activation of mitogen-activated protein kinase and nuclear factor kappa-light-chain-enhancer of activated B cells. In this study, we used a mouse model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropypridine (MPTP; a neurotoxin)-induced Parkinson’s disease (PD) to demonstrate the protective effect of FCE on dopamine neurons in the substantia nigra pars compacta (SNpc). Locomotor behavior and tyrosine hydroxylase immunohistochemical staining indicated that FCE significantly inhibited MPTP-induced dopaminergic cell loss in the SNpc. Glial fibrillary acidic protein immunohistochemistry and quantitative polymerase chain reaction analysis revealed that astroglial activation and tumor necrosis factor alpha, inducible nitric oxide synthase, and interleukin 1 beta production were significantly inhibited by FCE. The expressions of brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor, and nerve growth factor were markedly increased by FCE action against MPTP-induced toxicity. FCE showed a neuroprotective effect in a MPTP-induced PD model, which might be correlated with anti-inflammation and the stimulation of neurotrophic factors.

Nesfatin-1 protects dopaminergic neurons against MPP+/MPTP-induced neurotoxicity through the C-Raf\u2013ERK1/2-dependent anti-apoptotic pathway

Several brain-gut peptides have been reported to have a close relationship with the central dopaminergic system; one such brain-gut peptide is nesfatin-1. Nesfatin-1 is a satiety peptide that is predominantly secreted by X/A-like endocrine cells in the gastric glands, where ghrelin is also secreted. We previously reported that ghrelin exerted neuroprotective effects on nigral dopaminergic neurons, which implied a role for ghrelin in Parkinson’s disease (PD). In the present study, we aim to clarify whether nesfatin-1 has similar effects on dopaminergic neurons both in vivo and in vitro. We show that nesfatin-1 attenuates the loss of nigral dopaminergic neurons in the 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. In addition, nesfatin-1 antagonized 1-methyl-4-phenylpyridillium ion (MPP+)-induced toxicity by restoring mitochondrial function, inhibiting cytochrome C release and preventing caspase-3 activation in MPP+-treated MES23.5 dopaminergic cells. These neuroprotective effects could be abolished by selective inhibition of C-Raf and the extracellular signal-regulated protein kinase 1/2 (ERK1/2). Our data suggest that C-Raf-ERK1/2, which is involved in an anti-apoptotic pathway, is responsible for the neuroprotective effects of nesfatin-1 in the context of MPTP-induced toxicity. These results imply that nesfatin-1 might have therapeutic potential for PD.

14-3-3 inhibition promotes dopaminergic neuron loss and 14-3-3θ overexpression promotes recovery in the MPTP mouse model of Parkinson’s disease

14-3-3s are a highly conserved protein family that plays important roles in cell survival and interact with several proteins implicated in Parkinson’s disease (PD). Disruption of 14-3-3 expression and function has been implicated in the pathogenesis of PD. We have previously shown that increasing the expression level of 14-3-3θ is protective against rotenone and 1-methyl-4-phenylpyridinium (MPP+) in cultured cells. Here, we extend our studies to examine the effects of 14-3-3s in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We first investigated whether targeted nigral 14-3-3θ overexpression mediated by adeno-associated virus offers neuroprotection against MPTP-induced toxicity. 14-3-3θ overexpression using this approach did not reduce MPTP-induced dopaminergic cell loss in the substantia nigra nor the depletion of dopamine (DA) and its metabolites in the striatum at three weeks after MPTP administration. However, 14-3-3θ-overexpressing mice showed a later partial recovery in striatal DA metabolites at eight weeks after MPTP administration compared to controls, suggesting that 14-3-3θ overexpression may help in the functional recovery of those dopaminergic neurons that survive. Conversely, we investigated whether disrupting 14-3-3 function in transgenic mice expressing the pan 14-3-3 inhibitor difopein exacerbates MPTP-induced toxicity. We found that difopein expression promoted dopaminergic cell loss in response to MPTP treatment. Together, these findings suggest that 14-3-3θ overexpression promotes recovery of DA metabolites whereas 14-3-3 inhibition exacerbates neuron loss in the MPTP mouse model of PD.

Glucocerebrosidase deficiency and mitochondrial impairment in experimental Parkinson disease

Gaucher disease is an autosomal recessive disease, caused by a lack or functional deficiency of the lysosomal enzyme, glucocerebrosidase (GCase). Recently, mutations in the glucocerebrosidase gene (GBA) have been associated with Parkinson's disease (PD) and GBA mutations are now considered the most important genetic vulnerability factor for PD. In this study, we have investigated (i) in vivo whether inhibition of the enzyme glucosylceramide synthase by miglustat may protect C57Bl/6 mice against subchronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication and (ii) in vitro whether a decrease of GCase activity may render dopaminergic neurons susceptible to MPP+ (1-methyl-4-phenylpyridinium) or alpha-synuclein (α-Syn) toxicity and amenable to miglustat treatment. We could demonstrate that reduction of glucocerebroside by inhibition of glucosylceramide synthase partially protects mice against MPTP-induced toxicity. Conversely, we could show that inhibition of GCase activity with conduritol-B-epoxide (CBE) enhances both α-Syn and MPP+ induced toxicity in vitro. However, only CBE-induced enhancement of MPP+ toxicity could be reversed by miglustat. Moreover, we were unable to reveal any alterations of complex I activity or cell respiration upon treatment with either CBE or miglustat. Our findings suggest that the reduction of GCase activity rather than an accumulation of glucocerebroside increases aSyn toxicity.