Differential protein expression in substantia nigra induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine in a mouse model of chronic Parkinson’s disease

BACKGROUND： To date, a complete protein expression profile of the midbrain substantia nigra in a mouse model of chronic Parkinson＇s disease, induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine （MPTP）, does not exist. In addition, there are no reports of analysis of differential protein expression. OBJECTIVE： To separate and evaluate MPTP-induced differential protein expression through the use of proteomics in the substantia nigra of a mouse model of chronic Parkinson＇s disease. DESIGN： Randomized controlled animal study. SETTING： Department of Neurology, the First Affiliated Hospital, Chongqing Medical University. MATERIALS： Sixteen 8-10-week old, healthy, male, C57BL mice, weighing 20-25 g, and of clean grade, were provided by the Experimental Animal Center of Chongqing Medical University. The experimental animals were disposed according to ethical criteria. MPTP was provided by Sigma Company, USA; Pdquest 2D image analysis software and gelatum/irradiance image analysis system （ChemiDoc XRS） by Bio-Rad, USA; and Voyager DE-PROMALD1-TOF-MS mass spectroscopy analyzer by AB1 Company, USA. METHODS： This study was performed in Chongqing Neurological Laboratory between November 2006 and July 2007. Mice were randomly divided into model and control groups, with 8 mice in each group. Mice in the model group were received a subcutaneous injection of MPTP （25 mg＆g）, twice a week, for five successive weeks, to establish a chronic Parkinson＇s disease model. Mice in the control group received the same volume of a subcutaneous saline injection at the same time points. Mice were sacrificed by anesthesia to rapidly obtain the midbrain for protein separation of the substantia nigra. MAIN OUTCOME MEASURES： （1） 2-ED handbook （Bio-Rad Company） was referenced for two-dimensional electrophoresis, （2） PDQUEST8,0 analytical electrophoresis pattern was adopted to evaluate differential protein expression. （3） Peptide mass finger print map and data were retrieved on http：//www.prospector.ucsf.edu to compare differential substantia nigral protein expression in the two groups. RESULTS： Two-dimensional gel electrophoresis of substantia nigra tissue indicated that there were 33 differential protein expressions between the two groups. Three new proteins were evaluated, including α -enolase, which exhibited regulated expression, tumor necrosis factor ligand superfamily member 4, and cyclin-dependent kinase inhibitor 1B. CONCLUSION： There are three proteins that exhibit differential expression in the substantia nigra- α -enolase, tumor necrosis factor ligand superfamily member 4, and cyclin-dependent kinase inhibitor 1B.

THE EFFECTS OF DEPRENYL AND 1-METHYL-4-PHENYL-1, 2, 3, 6-TETRAHYDROPYRIDINE (MPTP) ON 2-DEOXYGLUCOSE UPTAKE IN THE MOUSE BRAIN

~3H-2-deoxyglucose (2-DG) autoradiographic technique was used to study the ef feets of a monoamine-oxidase-B (MAO-B) inhibitor deprenyl and the neurotoxin Ⅰ-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) on 2-DG uptake in the mouse brain. Following MPTP intoxication, 2-DG uptake was increased in the substantia nigra and lo(?)us ceruleus. At the same time, obvious abnormal behavior of the animals was induced. In the mice pretreated with deprenyl, 2-DG uptake was similar to that of control animal. Ab normal behavior. though present, was significantly milder than in mice given MPTP alone. It is concluded that MPTP interferes with the glucose metabolism in the substantia nigra and locus ceruleus and induces remarkable abnormal behavioral syndrome of mice. These deleterious effects can be blocked by pretreatment with deprenyl.

Establishing motor disorder mouse models of Parkinson disease Comparison of 6-hydroxydompamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

At present, pathogenesis and mechanism of Parkinson disease （PD） are still unclear. Animal models of PD are essential tools in studies on etiology and therapy and should mimic the chronic pathological process, histological characteristics and motor behavior dysfunction. In recent years, transgenic mice have been widely utilized to study the mechanism of PD, and it has become imperative that a PD mouse model of motor behavioral dysfunction be established. OBJECTIVE： To compare the behavioral and histochemical characters of two neurotoxic mice model induced with 6-hydroxydopamine （6-OHDA） or 1-methyl-4-phenyl-1, 2, 3, 6 -tetrahydropyridine （MPTP）, and a better method to mimic Parkinson disease will be found out. DESIGN： Parallel experiment. SETTING： Laboratory of Molecular Genetics, Department of Medical Genetics, Shanghai Jiao Tohg University. MATERIALS： Sixty 129Sv/C57BL6J male wild mice, SPF grade, 8 - 12 weeks old, weighing 20 - 25 g, were provided by Experimental Animal Center, Shanghai Jiao Tong University. All the surgery operation was performed according to the rules of Shanghai Jiaotong University Animal Committee. METHODS： The experiment was carried out in the Laboratory of Molecular Genetics （National Key Laboratory）, Department of Medical Genetics, Shanghai Jiao Ttong University from March to August 2006. ①Thirty-two male mice were randomly divided into control group and drug treatment group with 16 mice in each group. Surgery was carried out and 6-OHDA was administrated to substantia nigra pars compacta （SNpc） and nigra-striatum pathway according to the different parameters with intoxication apparatus. Saline was injected to the other 16 mice according to the same paradigm. 1 mg/kg apomorphine was injected intraperitoneally 2 weeks later after surgery to induce the imbalanced rotation behavior for 40 minutes. ②Twenty-eight mice were randomly divided into 4 groups with 7 in each group, including low-dose, moderate-dose, high-dose groups and negative control group. Then, mice in the drug treatment group were injected intraperitoneally with 5, 10 and 15 mg/kg MPTP for 9 successive days. In addition, mice in the control group were injected with the same volume of saline for 9 days. Pole test and stride length test were utilized to detect coordinative behavioral dysfunction. Mice were sacrificed 20 days after MPTP treatment, and histochemical staining of tyrosine hydroxynase （TH） was used to observe the loss of dopaminergic neuron in SNpc. MAIN OUTCOME MEASURES： ① Success ratio of each model establishment method; ② inducible asymmetric cycle behavior test 2 weeks after 6-OHDA injection; ③behavioral dysfunction in pole test and stride length, morphological changes in brain tissue. RESULTS： Totally sixty mice were used in this experiment and 3 mice were excluded because of the hypersensitivity or the clumsy reaction in motor behavioral detection before MPTP treatment, therefore, data was analyzed with the rest 57 mice. ① Lethal ratio： Three out of 16 mice died in striatum injection group and 5 out of 16 mice died in nigro-striatal pathway group. No mouse died in MPTP treatment groups. ② Locomotor behavior： No dysfunction of locomotor was found in 6-OHDA treatment groups. However, several motor behavioral dysfunction were start to present at the 4th day of MPTP injection. ③ Asymmetric cycle behavior： No asymmetric cycle was induced successfully two weeks after 6-OHDA surgery. Mice show hypersensitive behavior 10 minutes after apomorphine injection, which lasted for about 20 minutes. ④ Pole test： From the 4^th day of MPTP treatment, mice started to display coordinate dysfunction, such as climbing down along the pole in spiral, moving slowly with hesitation. Some mice could not grab the pole and slide down along the pole at 4th day post injection. Comparing with 0 dose control group, all the threedrug treatment groups show significant different dysfunction from the 4th day to the 20th day post injection （P 〈 0.01）. ⑤ stride length test： Mice＇s stride length decreased, when treated with MPTP, and the mice in the high dose group displayed obviously. ⑥ Dopaminergic neuron stained with TH in nigra pars compacta： The results indicated that administrated MPTP （from low dose to high dose） by intraperitoneal cause chronic lesions on the dopaminergic neuron in the SNpc. CONCLUSION： PD mice models induced with 6-OHDA show high mortality ratio and no asymmetric cycle was found after apomorphine injection. However, injection of MPTP intraperitoneally can simulate the chronic pathway of PD, typical histological changes are found and stable motor behavioral dysfunctions are displayed.

MADOPAR-INDUCED DYSKINESIA IN 1-METHYL-4-PHENYL-1,2,3,6-TETRAHYDROPYRIDINE (MPTP) HEMIPARKINSONIAN MONKEY MODEL

Infusion of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) into the right common carotid artery produced hemiparkinsonian syndrome on contralateral limbs in 5 rhesus monkeys. The hemiparkinsonian syndrome produced responded to madopar medication and the circling motion changed from toward the MPTP-treated side to away from the MPTP-treated side. Long term use of madopar developed a peak-dose dyskinesia of the face and limbs at the contralateral side. The toxic effect of MPTP was confirmed biochemically by reduction of nigrostriatal DA and histologically by degeneration of nigral neurons on the MPTP-treated side. It is concluded that this hemiparkinsonian monkey model will be of value in the elucidation of the neural mechanism underlying L-DOPA or DA agonists induced dyskinesia in Parkinson’s disease and in the search for newer methods of treatment which would produce less dyskinesia.

Prenatal and Postnatal Exposures to 1-Methyl-4-phenyl-1,2,3,6-tetra Hydropyridine (MPTP) Impaired Mouse Midbrain Dopamine System and May Produce a Predisposing and Inducing Model for Parkinson’s Disease

Dopamine cell bodies in the substantia nigra of the midbrain and with their terminals projecting to the neostriatum form the nigrostriatum and these dopamine neurons degenerate in Parkinson’s disease (PD). Based on metabolic and func- tional specialization of the cell bodies versus the axon terminals, the level and disposition of dopamine, its metabolites and enzymes are different in both regions and are likely to be affected differently in PD. We examined changes in the midbrain dopamine system following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), to test the hypothesis that a predisposing/sensitization stage and a inducing/precipitating stage underlie PD. Pregnant mice were treated with a low dose of MPTP during gestation days 8 - 12 to model the predisposing/sensitization stage, by interrupting the fetal mid- brain dopamine system during its neurogenesis. For the inducing/precipitating stage, the 12-weeks offspring were ad- ministered MPTP. The prenatal-MPTP offspring appear normal, but midbrain dopamine, 3,4-di-hydroxy-phenyl-acetic- acid, 3-methoxytyramine, tyrosine-hydroxylase and L-aromatic-amino-acid-decarboxylase, were reduced by 49.6%, 48%, 54%, 20.9% and 25%. Postnatal-MPTP of 10, 20, 30 mg/kg administered to the prenatal-PBS vs prenatal-MPTP offspring reduced midbrain dopamine by 43.6%, 47.2%, 70.3% vs 85.4%, 89.1%, 95.2%;tyrosine-hydroxylase by 30%, 63%, 81% vs 30.7%, 70.4%, 91.4%;L-aromatic-amino-acid-decarboxylase by 0%, 2%, 40% vs 32%, 40%, 58%. The prenatal-MPTP may render the DA system sensitive by causing sub-threshold reduction of DA, its metabolites and en- zymes, enabling postnatal-MPTP to reduce dopamine above the 70% - 80% PD-inducing threshold. Thus, the study may produce a prenatal predisposing/sensitization and postnatal inducing/precipitation model of PD. It also indicates that some cases of PD may have a fetal basis, in which sub-threshold nigrostriatal impairments occur early in life and PD-symptoms are induced during aging by further insults to the dopaminergic system that would not cause PD symptoms in normal indi-viduals.

The transient receptor potential melastatin 2:a new therapeutical target for Parkinson's disease?

The transient receptor potential melastatin 2 is a calcium-permeable cation channel member of the TRP family. Also known as an oxidative stress-activated channel, the transient receptor potential melastatin 2 gating mechanism is dependent on reactive oxygen species. In pathological conditions, transient receptor potential melastatin 2 is overactivated, leading to a Ca~(2+) influx that alters cell homeostasis and promotes cell death. The role of transient receptor potential melastatin 2 in neurodegenerative diseases, including Alzheimer's disease and ischemia, has already been described and reviewed. However, data on transient receptor potential melastatin 2 involvement in Parkinson's disease pathology has emerged only in recent years and the issue lacks review studies that focus specifically on this topic. The present review aims to elucidate the role of the transient receptor potential melastatin 2 channel in Parkinson's disease by reviewing, summarizing, and discussing the in vitro, in vivo, and human studies published until August 2022. Here we describe fourteen studies that evaluated the transient receptor potential melastatin 2 channel in Parkinson's disease. The Parkinson's disease model used, transient receptor potential melastatin 2 antagonist and genetic approaches, and the main outcomes reported were discussed. The studies described transient receptor potential melastatin 2 activation and enhanced expression in different Parkinson's disease models. They also evidenced protective and restorative effects when using transient receptor potential melastatin 2 antagonists, knockout, or silencing. This review provides a literature overview and suggests where there is a need for more research. As a perspective point, this review shows evidence that supports transient receptor potential melastatin 2 as a pharmacological target for Parkinson's disease in the future.

Aspects on the Mechanism of the 1-Phenyl-1<i>H</i>-pyrazolo[3,4-<i>b</i>]quinoxaline Formation

Condensation of D-glucose, o-phenylenediamine and N,N-benzylphenylhydrazine hydrochloride (NNBPHH) in a one-pot reaction, or condensation of 2-(D-arabino-tetritol-1-yl) quinoxaline and NNBPHH, gave 3-(D-erythro-glycerol-1- yl)-1-phenyl-1H-pyrazolo[3,4-b]quinoxaline. The structure of the latter was determined by 1H NMR spectroscopy and by synthesis using phenylhydrazine hydrochloride instead of NNBPHH. Condensation of D-glucose and 4,5-dichloro-o-phenylenediamine gave 6,7-dichloro-2-(D-arabino-tetritol-1-yl)quinoxaline, which upon condensation with NNBPHH gave the corresponding 6,7-dichloro-3-(D-erythro-glycerol-1-yl)-1-phenyl-1H-pyrazolo[3,4-b]quinoxaline. The structure and mechanism of formation of these compounds are discussed.

Exendin-4 and linagliptin attenuate neuroinflammation in a mouse model of Parkinson's disease

Use of glucagon-like peptide-1 receptor agonist or dipeptidyl peptidase 4 inhibitor has been shown to lower the incidence of Parkinson's disease in patients with diabetes mellitus.Therefore,using these two treatments may help treat Parkinson's disease.To further investigate the mechanisms of action of these two compounds,we established a model of Parkinson's disease by treating mice with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and then subcutaneously injected them with the glucagon-like peptide-1 receptor agonist exendin-4 or the dipeptidyl peptidase 4 inhibitor linagliptin.We found that both exendin-4 and linagliptin reversed motor dysfunction,glial activation,and dopaminergic neuronal death in this model.In addition,both exendin-4 and linagliptin induced microglial polarization to the anti-inflammatory M2 phenotype and reduced pro-inflammatory cytokine secretion.Moreover,in vitro experiments showed that treatment with exendin-4 and linagliptin inhibited activation of the nucleotide-binding oligomerization domain-and leucine-rich-repeat-and pyrin-domaincontaining 3/caspase-1/interleukin-1βpathway and subsequent pyroptosis by decreasing the production of reactive oxygen species.These findings suggest that exendin-4 and linagliptin exert neuroprotective effects by attenuating neuroinflammation through regulation of microglial polarization and the nucleotidebinding oligomerization domain-and leucine-rich-repeat-and pyrin-domain-containing 3/caspase-1/interleukin-1βpathway in a mouse model of Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.Therefore,these two drugs may serve as novel anti-inflammatory treatments for Parkinson's disease.

Single-neuron neurodegeneration as a degenerative model for Parkinson’s disease

The positive effect of levodopa in the treatment of Parkinson’s disease,although it is limited in time and has severe side effects,has encouraged the scientific community to look for new drugs that can stop the neurodegenerative process or even regenerate the neuromelanin-containing dopaminergic nigrostriatal neurons.Successful preclinical studies with coenzyme Q10,mitoquinone,isradipine,nilotinib,TCH346,neurturin,zonisamide,deferiprone,prasinezumab,and cinpanemab prompted clinical trials.However,these failed and after more than 50 years levodopa continues to be the key drug in the treatment of the disease,despite its severe side effects after 4–6 years of chronic treatment.The lack of translated successful results obtained in preclinical investigations based on the use of neurotoxins that do not exist in the human body as new drugs for Parkinson’s disease treatment is a big problem.In our opinion,the cause of these failures lies in the experimental animal models involving neurotoxins that do not exist in the human body,such as 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine,that induce a very fast,massive and expansive neurodegenerative process,which contrasts with the extremely slow one of neuromelanin-containing dopaminergic neurons.The exceedingly slow progress of the neurodegenerative process of the nigrostriatal neurons in idiopathic Parkinson’s patients is due to(i)a degenerative model in which the neurotoxic effect of an endogenous neurotoxin affects a single neuron,(ii)a neurotoxic event that is not expansive and(iii)the fact that the neurotoxin that triggers the neurodegenerative process is produced inside the neuromelanin-containing dopaminergic neurons.The endogenous neurotoxin that fits this degenerative model involving one single neuron at a time is aminochrome,since it(i)is generated within neuromelanin-containing dopaminergic neurons,(ii)does not cause an expansive neurotoxic effect and(iii)triggers all the mechanisms involved in the neurodegenerative process of the nigrostriatal neurons in idiopathic Parkinson’s disease.In conclusion,based on the hypothesis that the neurodegenerative process of idiopathic Parkinson’s disease corresponds to a single-neuron neurodegeneration model,we must search for molecules that increase the expression of the neuroprotective enzymes DT-diaphorase and glutathione transferase M2-2.It has been observed that the activation of the Kelch-like ECH-associated protein 1/nuclear factor(erythroid-derived 2)-like 2 pathway is associated with the transcriptional activation of the DT-diaphorase and glutathione transferase genes.

An environmentally friendly synthesis of 1,4-dihydropyrano[2,3-c]pyrazole derivatives catalyzed by tungstate sulfuric acid

An efficient three-component synthesis of 6-amino-4-aryl-5-cyano-3-metriyl-1-phenyl-1,4-dihydropyrano[2,3-c]pyrazoles via a reaction between 3-methyl-1-phenyl-2-pyrazolin-5-one,aromatic aldehydes and malononitrile using tungstate sulfuric acid as a catalyst was described.Mild conditions,good to excellent yields,easily available catalyst and easy work-up are the key features of this method.

Isatin decreases Bax protein expression in the substantia nigra of a mouse model of Parkinson's disease

The present study observed the action of 1H-indole-2, 3-dione （isatin） on Bax protein expression in the substantia nigra of a Parkinson＇s disease animal model. Parkinson＇s disease-like behaviors were induced in C57BL/6J mice treated with 1-methyl-4-phenyl-1,2, 3, 6-tetrahydropyridine （MPTP） Bax protein expression was significantly reduced in isatin （100, 200 mg/kg）-pretreated mice. Results demonstrate that isatin plays a neuroprotective role in mice treated with MPTP by down-regulating Bax protein expression.

Astrocyte reactivity in related brain regions in a mouse model of MPTP-induced Parkinson's disease

BACKGROUND： Severe injury to dopaminergic neuronal cell bodies and their axon terminals in the substantia nigra pars compacta （SNC） has been observed in both Parkinson＇s disease （PD） patients or in 1-methy-4-phenyl-1,2,3,6-tetrahydropyrindine（MPTP）-induced PD animal models, but only slight injury occurs in the adjacent ventral tegmentat area （VTA）. The mechanisms underlying this selective injury remain poorly understood. OBJECTIVE： To comparatively observe astrocyte reactivity in the SNC, caudate putamen （CPu）, VTA, and frontal association cortex （FrA）. DESIGN, TIME AND SETTING： A cellular and molecular biology, randomized, controlled experiment was performed at the Institute of Neurobiology, Department of Human Anatomy, Medical School of Nantong University, between December 2006 and September 2008. MATERIALS： A total of 80 healthy adult male C57BL/6 mice were included in this study. MPTP was purchased from Sigma, USA. METHODS： Mice were randomly divided into a model group （n = 64） and a sham-operated group （n = 16）. PD was induced in the mice from the model group by intraperitoneal injection of 20 mg/kg MPTP, once every three hours, for a total of 4 times. MAIN OUTCOME MEASURES： Tyrosine hydroxylase （TH）-immunoreactive neurons and glial fibrillary acidic protein （GFAP）-immunoreactive astrocytes were examined by dual immunofluorescence labeling. GFAP-immunoreactive astrocytes in the CPu and FrA were determined by immunofluorescent staining. GFAP mRNA expression in the SNC, CPu, VTA, and FrA was detected using real-time polymerase chain reaction. TH protein levels in the TH-immunoreactive axon terminals of the CPu and FrA were detected by Western blotting. RESULTS： Numbers of TH-immunoreactive neurons in the SNC, and TH protein level in the CPu, markedly decreased （by approximately 68%） 1 day after MPTP injection, and gradually increased at 3 days. Simultaneously, astrocyte reactivity was strengthened, in particular at 7 days. However, after MPTP injection, decreases in the numbers of TH-immunoreactive neurons in the VTA, and TH protein levels in the FrA, were less apparent （approximately 15%）. Also, no obvious astrocyte reactivity was observed. CONCLUSION： In a mouse model of PD, astrocyte reactivity was apparent in the SNC and CPu, but not the VTA or FrA. In addition, astrocyte reactivity was greater in regions where injury to dopaminergic neurons was more severe.

A New Chromone Derivative from Stellera chamaejasme L

A new chromone derivative, 3-[1- (2, 4, 6-trihydroxyphenyl) 3-di-(4-hydroxyphenyl) 1-propanone-2-yl] 5,7-dihydroxy-8-di(4-hydroxyphenyl)methyl-4H-1-benzopyran-4-one, named as isomohsenone was isolated from the roots of Stellera chamaejasme L. together with known chamaechromone. Its structure was determined by the analysis of MS and NMR data, especially 2D NMR spectra.

Epigenetic mechanisms involved in the neuroprotective effect of scorpion extract in a Parkinson's disease murine model based on multi-omics approach

OBJECTIVE: To investigate whether scorpion extract elicits a neuroprotective effect in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-treated mice models, and the genes associated with the therapeutic effects using RNA sequencing(seq)analysis.METHODS: This study investigated the changes in interaction between messenger ribonucleic acid(m RNA) expression and deoxyribonucleic acid(DNA) methylation related to the protective effects of scorpion extracts, in the substantia nigra(SN)region of a MPTP-induced Parkinson's disease(PD)model.RESULTS: In this model, scorpion extracts attenuated the motor impairment as demonstrated by the rotarod and open field tests. Scorpion extracts consistently attenuated the decrease of tyrosine hydroxylase(TH) positive neural cells in the SN and striatum of mice. We profiled genomewide DNA methylation using Methyl-Seq and measured the transcriptome using RNA-Seq in murine SN in the following groups: vehicle-treated MPTP-induced PD mice and scorpion extracttreated MPTP-induced PD mice. In total, 13 479 differentially expressed genes were identified in association with the anti-PD effect of the scorpion extract, mainly in the promoter and coding regions.Among them, 47 were negatively correlated downregulated genes. Nineteen genes out of 47 downregulated genes were negatively correlated with the expression of the other 28 genes. Among these genes, SGSM1 was related to dopaminergic neurons including dopamine transporters, TH, dihydroxyphenylalanine decarboxylase, and dopamine D2 receptor.CONCLUSION: This study provides insights into the anti-parkinsonian effects of scorpion extract and reveals the epigenetic targets in its therapeutic mechanism.