| Parkinson’s disease (PD) is a neurodegenerative disease characterized by the loss of dopaminergici neurons in substantia nigra, the main clinical manifestations of which are tremor, rigidity, and bradykinesia. The incidence of PD in people whose ages were over 60 was 1%. Patients suffering PD lost the ability of voluntary movement and got cognitive deficit if severely, which give greatly bad effects to one’s life. Before emergence of clinical symptoms, dopaminergic neurons can heal strongly by itself. But, once the clinical manifestation of PD appears, almost 75% dopaminergic neurons have already lose and fail to recover. Nowadays, the exact pathogenesis and mechanism of PD are still under investigation. Medicines and methods which can overwhelm or inhibit the progress of PD are lacking. This severe situation makes people urge to explore the exact mechanism of PD and treatments which could overcome PD effectively. To study PD pathogenesis and treatment of concrete becomes an urgent research topic.Based on reports, the etiology of PD is closely associated with environment, hereditary, aging, cell apoptosis, autoimmunity, oxidative stress, excitatory neurotoxin and so on. Above, the protein named tau which is very important to the growth and development drew more and more attention of investigators. Tau was found in cytoplasm of neurons and abundant in the growth point of axons and dendrite. Since tau plays a critical role in the upgrowth, morphology, material transportation, signal transduction, it becomes a hotspot in the research field of neurodegenerative diseases. Abnormal modification, such as hyperphosphorylation, glycosylation, could make tau or other proteins related to neurodegenerative diseases fold and aggregate mistakenly, which cause dysfunction or death of cells. So far, multiple signal transduction pathways were found to lead to apoptosis of dopaminergic neurons in substantia nigra, such as c-Jun activation, p53 activation, cell cycle reactivation, GSK-3β activation, CDK 5 activation, Bcl-2 expression. Tau is the common substrate of some key kinases in nearly all above signal pathway, which showed hyper phosphorylation in PD patients and animal models. For this reason, regulation of tau’s function is hopeful to provide new methods to treat PD.The function of tau is mainly regulated by its phosphorylation, so hyperphosphorylation of tau is closely related to the apoptosis of neurons. If tau is hyperphosphorylated, its functions of stabilizing microtubules and supporting neurons’ growth will meet a failure. Multiple kinases are involved in phosphorylation of tau in vivo, but the most important kinases are two:glycogen syntheses kinase 3β (GSK-3β) and Cyclin-dependent protein kinase 5 (CDK5). Therefore, it is possible that inhibiting the hyper phosphorylation of tau via regulating the two kinases’ activity can prevent the dopaminergic neurons from apoptosis. This had already been verified in the preliminary study of our research. In a PD vitro model,6-Hydroxy-lH-indazole could protect SH-SY5Y cells from apoptosis induced by MPP+. Because the compound can down-regulate hyperphosphorylation of tau by inhibiting the activity of GSK-3β and CDK5. Though, the experiments in vitro had already demonstrated that 6-Hydroxy-lH-indazole could down-regulated the phosphorylation of tau via inhibiting the activity of GSK-3β and CDK5 to protect dopaminergic neurons from apoptosis, however, whether it has the same effects in vivo or not is unknow yet. In this study, the PD animal model was established by high dose administration of MPTP to investigate whether 6-Hydroxy-1H-indazole have the effect of protecting dopaminergic neurons, and whether the mechanism is decreasing the hyper phosphorylation of tau.12 weeks old adult male C57BL/6 mice were used and divided into 4 groups with each group of 10:group treated with normal saline, group treated with MPTP, group treated with MPTP combined low dose of 6-Hydroxy-1H-indazole, group treated with MPTP combined high dose of 6-Hydroxy-1H-indazole. Once the mice were given MPTP, they would develop the PD symptoms of tremor. The incubation period and the maintenance period of the symptoms were recorded. After several days’ administration, western blotting, ABC staining procedure, fluorescent double-labeling experiment, behavioral experiment and high performance liquid chromatography were respectively utilized to observe the expression of TH, the survival situation of dopaminergic neurons, the phosphorylation of tau, the injure of behavior and the concentration of dopamine in the striatum respectively. The results of the experiments above had demonstrated that 6-Hydroxy-1H-indazole can inhibit the hyperphosphorylation of tau to protect dopaminergic neurons in the vivo in C57BL/6 mouse.Methods1. The establishment of the PD mouse model via intraperitoneal injection of MPTP and 6-Hydroxy-1H-indazole12 weeks old adult male C57BL/6 mice were randomly divided into 4 groups with each group of 10:group treated with normal saline (control group), group treated with MPTP, group treated with MPTP combined 2 mg/kg of 6-Hydroxy-1H-indazole (low-dose group), group treated with MPTP combined 4 mg/kg of 6-Hydroxy-1H-indazole (high-dose group). Control group received normal saline with the same volume to the other groups. MPTP group was given vehicle at day 1 (i.p.) and followed by MPTP 30mg/kg/day (i.p.) between day 2 and 6. The rest two groups were respectively treated with 6-Hydroxy-lH-indazole 2 mg/kg/day and 4 mg/kg/day (i.p.) 1 day before the initiation of MPTP administration (30 mg/kg), and in the following 5 days 6-Hydroxy-lH-indazole was daily injected (i.p.) half an hour before MPTP treatment.2. The observation of the incubation period and the maintenance period of PD symptomsFrom the second MPTP injection, the PD symptoms’incubation period and maintenance period of each mouse were observed and recorded. The incubation period:the period between the MPTP injection and the appearance of PD symptoms. The maintenance period:the time from the beginning of PD symptoms to complete obliteration. The comparisons of the incubation period and maintenance period among groups were respectively made to investigate whether 6-Hydroxy-lH-indazole could alleviate the mouse’s PD symptoms.3. Determination of survival dopaminergic neurons in substantia nigra of mouseOn day 7 after the final injection of MPTP, brains were serially sectioned (35μm) through the entire midbrain. Sections were processed by 0.3% H2O2 in TBS for 30 min after washing by TBS/Triton, blocked by 3% goat serum in TBS/Triton for 1 hour after washing by TBS/Triton 2 times, incubated with anti-TH antibodies overnight at 4℃. Next day, after washing 2 times, sections were incubated with goat anti-rabbit secondary antibody for 2 hours and ABC reagent for 1 hour. With times’ washing in TBS/Triton or TBS, sections were dying in DAB reagent for minutes and pasted on glass slides overnight at RT. Next day, sections were successively put into 50%,70%,80%,90%,95%,100% ethyl alcohol for 2 minutes and ethyl alcohol with xylene (1:1), xylene for 3 minutes. In the end, sections were mounted with neutral resins and kept at RT.4. Detection of TH’s amount in midbrain of mouseOn day 7 after the final injection of MPTP, midbrains were rapidly separated after mice were killed by cervical dislocation. Midbrains were weighed, homogenized by lysis buffer at 500μl/50mg, sonicated, centrifuged 12000 rpm for 30 minutes at 4 ℃. The supernatant were collected, measured by BCA method and kept in -20℃ for western blotting.5. Behavioral testA pole test was performed in the morning on day 10 after the final MPTP to detect impairment of limb movement. Briefly, a cork ball was fixed to the top of a vertical pole. The pole was doubly wrapped with gauze to prevent slipping. The mouse was placed on the cork ball. The time required for the mouse to turn downward on the ball, to climb down the upper half of the pole, and climb down the lower half of the pole were recorded. Finishing performance within 3,6 and over 6 s were scored as 3,2 and 1, respectively. The results were expressed as the total of score.Traction test:hanged from a horizontal wire by its fore paws, a mouse was scored according to how many hind paws it used to grip the wire. Gripping the wire with both hind paws, one hid paws, and neither hind paws were scored 3,2, and 1, respectively. The score of mice in each group were statistically analyzed.6. Determination of the concentration of dopamine in striatum by HPLCStriatum were separated after the behavioral tests and kept at -70℃ until analyzed. When detected, samples were homogenized in 0.5 ml extracting solution including perchloric acid, and centrifuged at 18000 rpm and 4℃ for 20 minutes. The supernatants were collected.25-100μl of each supernatant was analyzed by HPLC. Chromatographic condition:510 high-pressure pump (Waters),7125 sample injector, HP1049A electrochemical detector, BDS C18 chromatographic column with size at 4.6*250 mm,5μm. Buffer solution:3 mM sodium heptane-1-sulphonate,100 mM sodium acetate,85 mM citric acid,0.2 EDTA, PH 4.0. Mobile phase:90% buffer solution with 10% methyl alcohol. Rate of flow:1.0 ml/min, column temperature:40 ℃. Qualitative analysis was processed through the comparison of the retention time between sample and standard. Quantitative method:DHBA was used as internal standard substance, the ratio of peak area between samples and the substance was calculated to quantitative analysis.7. Detection tau phosphorylation in dopaminergic neurons in the substantia nigra of mouseMice were killed after the third MPTP injection. The brains were cut into 10 μm coronal section to facilitate double-labeling experiments. Rabbit anti-TH and goat anti-p-tau antibodies were respectively used as primary antibodies, rabbit anti-rabbit IgG (FITC labeled) and anti-goat IgG (carbocyanine-3 labeled) were used as secondary antibodies. Sections were put into 24-pore plate and washed by TBS/Triton once, then blocked by 3% donkey serum in TBS/Triton for 1 hour, incubated by anti-TH in 3% BSA dissolved in TBS overnight at 4℃. On next day, sections were washed by TBS/Triton for two times and incubated with anti-p-tau in 3% BSA dissolved in TBS for 2 hours at 37℃. After the incubation of the two primary antibodies above, sections were washed by TBS/Triton and TBS successively, incubated with the mixture of secondary antibodies in 1% BSA dissolved in TBS for 1 hour at RT and kept out of the light from now on. Next, sections were pasted on glass slide after two-time wash. In the end, sections were analyzed by fluorescence microscope.8. Statistical analysisData were analyzed using SPSS 13.0 statistical software and expressed as the mean±S.E.M. Statistical analysis was completed using a one-way ANOVA with LSD’s t test for multiple comparisons of the means. Differences were considered to be statistically significant with P<0.05.Results1.6-Hydroxy-1H-indazole alleviates the MPTP-induced symptoms of PDAfter administration of MPTP, mice in MPTP group rapidly developed more severe symptoms of PD as shown in the shorter incubation period (1.89±0.08 min) and longer maintenance period (25.58 ± 0.51 min). While mice treated with 6-Hydroxy-lH-indazole (2 mg/kg and 4 mg/kg) combined MPTP developed milder symptoms of PD, indicated by the delayed incubation period (3.97±0.15 min; 3.51± 0.09 min) and shorter maintenance period (16.85±0.72 min; 16.48±0.45 min) of symptom. Mice in vehicle control group didn’t experience the symptoms above.2.6-Hydroxy-1H-indazole protected dopaminergic neurons from apoptosis induced by MPTPPreviously we have shown that 6-Hydroxy-lH-indazole protects SHSY5Y cells from the toxicity of MPP+. A significant loss of TH+ neurons was observed in mice treated with MPTP compared to mice in control group, which could be evidenced by the data TH+ neurons of MPTP group was about 36.65% of control. In contrast, treatment of 6-Hydroxy-lH-indazole at dose of 2 mg/kg and 4 mg/kg combined MPTP markedly suppressed the loss of TH+ neurons, the TH-positive neurons of two groups were respectively 78.61% and 93.63% of control group, both were significantly higher than MPTP group. Even, in the higher dose, 6-Hydroxy-lH-indazole almost completely protected TH+ neurons from apoptosis. As shown in the results,6-Hydroxy-lH-indazole prevented dopaminergic neurons from apoptosis induced by MPTP.3.6-Hydroxy-lH-indazole attenuated the depletion of TH induced by MPTPIndicated by data, the level of TH was measured by Western blot in this experiment. Compared with mice in control group, level of TH was significantly reduced in MPTP group in which the TH-level was about 45% of control. However, the depletion of TH induced by MPTP was prevented by 6-Hydroxy-lH-indazole as shown in the darker bands of two 6-Hydroxy-lH-indazole combined MPTP treated groups. What’s more, the level of TH was recovered more significantly with the treatment of higher dose of 6-Hydroxy-lH-indazole in which the TH-level was about 88% of control.4.6-Hydroxy-1H-indazole ameliorated the behavioral damage of MPTP treated miceGiven 6-Hydroxy-lH-indazole combined MPTP, we investigated that limb movement of mice was improved when comparing to mice of MPTP group.In pole test, total time required for mice in MPTP-treated group to turn downward and climb from the top of the pole to the ground was remarkably longer than mice in groups treated with 6-Hydroxy-lH-indazole combined MPTP as shown in the score:mice in MPTP groups got 5.1 points, while both of mice in 6-Hydroxy-1H-indazole 2 mg/kg and 4 mg/kg combine MPTP treated groups got 7.1 points.In traction test, the vehicle-treated mice in control group gripped the wire by all four paws, while in MPTP group mice gripped the wire only by their fore paws, both of which could be indicated by the score:mice in control group got 2.6 points and mice in MPTP group got 1.7 points. However, mice treated with 6-Hydroxy-lH-indazole combined MPTP gripped the wire by one or two hind paws, and mice treated with dose of 2 mg/kg and 4 mg/kg got 2.4 points and 2.2 points respectively, both of them were significantly higher than the score of MPTP group. As shown in the score, the comparison suggested that 6-Hydroxy-lH-indazole alleviated the behavioral damage induced by MPTP.5.6-Hydroxy-lH-indazole increased the striatal dopamine concentration following MPTP exposureAs dopamine level is a mark of dopaminergic synaptic function, striatal dopamine concentrations were measured by HPLC. Pointed out by data, the striatal dopamine concentration of mice treated with MPTP (2.75±0.16 ng/mg) was markedly decreased, while the DA concentration in control group was 12.54±0.27 ng/mg. However, with the treatment of 6-Hydroxy-lH-indazole, the reduction was significantly attenuated in mice treated with 6-Hydroxy-lH-indazole combined MPTP, shown by data that the concentration of DA in groups with dose of 6-Hydroxy-lH-indazole 2 mg/kg and 4 mg/kg were 4.63±0.56 ng/mg and 4.14± 0.13 ng/mg, both of which were significantly higher than concentration of MPTP group.6.6-Hydroxy-lH-indazole decreased tau hyperphosphorylation induced by MPTP in TH+ cells in SNcThe level of tau phosphorylation at Ser396 in dopaminergic neurons was assessed by immunohistochemistry analysis. Dopaminergic neurons were identified by co-labeling of TH. A significant increase in the level of p-tau was observed in the SNc of the mice treated with MPTP [52.19%] as compared to the control SNc [17.58%]. To test the effects of 6-Hydroxy-lH-indazole on the phosphorylation of Tau in vivo,6-Hydroxy-lH-indazole of 2 mg/kg and 4 mg/kg were given to the mouse half an hour before MPTP treatment. The phosphorylation of Tau in TH+ neurons was dramatically decreased to 21.17% and 17.85% respectively. Thus 6-Hydroxy-lH-indazole protected dopaminergic neurons from MPTP toxicity and inhibited of hyperphosphorylation of tau at Ser396.Conclusions1. Established stable PD animal model using medication of MPTP in large dose: MPTP injection caused phosphorylation of tau in a short time. It lead to dopaminergic neurons apoptosis, TH-positive cells in nigra loss, striatal dopamine concentrations decrease and limbs coordination disorder;2.6-Hydroxy-1H-indazole can decrease the phosphorylation of tau induced by MPTP in nigral neurons, reduced neuronal apoptosis, raised the number of survival dopaminergic neurons, increased striatal dopamine concentrations and improved behavioral changes of PD mice;3. Our results suggested a new strategy for researching anti-PD drugs, which was inhibition of tau phosphorylation of medicine effect. |
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