Neuroprotective Effect And Mechanism Of TrkB Receptor Agonist On The Animal Models Of Parkinson’s Disease

Section 1 Small molecule TrkB agonist deoxygedunin protects nigrostriatal dopaminergic neurons from MPTP induced neurotoxicity in rodentsObjective The major purpose of this study was to study the neuroprotective effect of deoxygedunin in MPTP-induced mice model of Parkinson’s disease.Method Thirty-six C57/BL6 mice were randomly divided into three groups:normal group, PD group and intervention group. Mice were given deoxygedunin 5 mg/kg (i.p.) for 2 weeks and administrated with MPTP twice at the dose of 20mg/kg (i.p.) on day 7. Pole test was used to assess the motor ability of the PD models. TH immunostaining was performed to evaluate MPTP induced neurotoxicity on SN dopaminergic neurons and the DA terminal in striatum. Co-expression of TH and phospho-TrkB (Y816) in the substantia nigra of the MPTP neurotoxicity mouse were detected by immunofuorescent staining. RT-PCR was used to examine the expression level of BDNF, a-synuclein and TrkB mRNA. Western blotting analysis was used to examine the activation of TrkB receptor and its downstream signaling pathways such as p-TrkB/TrkB、p-MAPK/MAPK、 p-Akt (S473)/Akt and Bcl-xl.Results The weigh in MPTP-treated mice were much lower than that of normal group for the neurotoxicity of MPTP (p<0.05), but pre-treatment with deoxygedunin ameliorated the loss of weight. MPTP treated mice without deoxygedunin treatment had longer time to orient downward (T-turn) and to descend (TLA) than that of normal animals, indicating impaired motor function (P<0.05). Pretreatment with deoxygedunin decreased Tturn and TLA compared to the MPTP group (P<0.05). Positive cell counting of TH immunostaining revealed that the number of TH positive neurons in MPTP group was significantly reduced compared with normal control and deoxygedunin group (P<0.05). There was no significant difference of the BDNF and a-synuclein mRNA between the three groups, but the level of TrkB mRNA in deoxygedunin group was increased compared with other two groups. TrkB receptor was more prominently phosphorylated in deoxygedunin group than the MPTP and normal groups, so were the downstream pathways involving MAPK and PI3K/Akt pathways, and there was no significant difference in the expression of BDNF in the SN between each group. As expected, we found that deoxygedunin protected neurons from apoptosis by up-regulating the Bcl-xl expression and suppressing caspase 3 activation.Conclusions The results revealed that deoxygedunin improved the behavioral performance of MPTP-induced PD model and reduced dopaminergic neurons loss in SN, associated with the activation of TrkB receptors and its two major signaling cascades involving mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K).Section 2 Cellular co-localization of tau fragment N368 and alpha-synuclein in three animal models of Parkinson’s diseaseObjective Tau fragment N368, cleavaged by AEP at N368, has been shown to aggregate and strongly trigger neurodegeneration. The goal of this study was to examine the distribution of a novel tauopathology and its interaction with alpha-synuclein in three animal models of Parkinson’s disease (PD).Methods Three animal models of PD were evaluated:1 mg/kg rotenone once daily for 50 days in C57/BL6 mice,1.5mg/kg rotenone once daily for 35 days and 25mg/kg MPTP twice one week for 5 weeks were respectively given subcutaneously to induce Parkinson’s disease models. TH immunostaining was performed to evaluate MPTP induced neurotoxicity on SN dopaminergic neurons. Immunohistochemical staining was used to investigate the distribution of tau fragment N368 and alpha-synuclein. Double-immunofluorescence study was performed to examine the co-localization of tau fragment N368 and alpa-synuclein of the different areas in the brain of three PD animal models. Western blot was used to detect the expression levels of tau fragment N368 and alpha-synuclein ser129 in substantia nigra, striatum and hippocampus of the three PD animal models.Results Decreased numbers of TH positive neurons in the substantia nigra of these three PD animal models were demonstrated. Double-label immunofluorescence microscopy demonstrated cellular co-localization of tau fragment N368 and alpha-synuclein in striatum and SN of the rotenone-induced PD animal models except the hippocampus and cortex compared to the control group:No changes of a-synuclein and tau N368 in the CA1 area of hippocampus were found in the rotenone-induced PD mice, but along with the increased level of tau N368 in the cortex compared with the control group. The expression of tau N368 increased in the CA1 area of the hippocampus and substantia nigra of the MPTP-induced PD model, and there was no difference in the expression of a-synuclein at the same site. There was no significant difference on the both expressions of tauN368 and a-synuclein in the cortex of the above animal model. Immunohistochemical staining showed high expression level of a-synuclein and tau N368 in striatum compared with control group in the rotenone-induced PD rat model. The expression levels of p-a-synuclein in the substantia nigra and hippocampus of PD rat model increased, and there was on significant difference on the level of tau N368. Western blot analyses revealed a significant high expression of tau fragment N368, alpha-synuclein serl29 and AEP in striatum, while there was no consistent expression in hippocampus and substantia nigra between the control and PD group. The levels of tau N368 in the hippocampus and substantia nigra increased in the MPTP-induced PD mice model, with no changes of p-a-synuclein (S129) on the same site. There was no significant difference of p-a-synuclein(S129) and tau N368 in the hippocampus between the rotenone-induced PD mice model and the control group. The expression of p-a-synuclein(S129) in the hippocampus of rotenone-induced PD rat models increased, with no changes of tau N368 in the same site.Conclusions The phenomenon of tau fragment N368 produced by activated-AEP was demonstrated in both striatum and substantia nigra of the PD animal models compared with control group. The co-localization between tau fragment N368 and alpha-synuclein in the substantia nigra and striatum of PD animal models indicated that activated AEP might play an important role in the pathogenesis of PD.Section 3 7,8-dihydroxyflavone protects nigrostriatal dopaminergic neurons from rotenone-induced neurotoxicity in rodents by regulating the expression of AEP and its trunction effectObjective The major purpose of this study was to investigate the neuroprotective effect of 7,8-dihydroxyflavone in rotenone-induced mice model of Parkinson’s disease.Method Thirty-six SD rats were randomly divided into three groups:control group, rotenone group and intervention group. Rats in the intervention group were given 7,8-DHF 5 mg/kg/day (i.p.), and animals in the both intervention and rotenone groups were administrated with rotenone at the dose of 2mg/kg/day (i.h.) for 5 weeks. Open field test was used to assess the motor ability of the animals in this study. TH immunostaining was performed to evaluate rotenone-induced neurotoxicity on SN dopaminergic neurons and the DA terminals in the striatum. Immunohistochemical method was used to evaluate the phosphorylation of TrkB receptor. Western blotting analysis was used to examine the expression of TH、BDNF、p-TrkB/TrkB‘ p-MAPK/MAPK’p-Akt (S473)/Akt、AEP、p-a synuclein(Ser129)、a-synuclein、 p-tau (Ser396) and tauN368 in the substantia nigra and striatum.Results The rotenone-induce PD model displayed worse locomotor activity in the open field test, and the intervention of 7,8-DHF could ameliorate the motor dysfunction of the PD animal model. Compared to the rotenone group, treatment with 7,8-DHF significantly attenuated the loss of dopaminergic neurons in SNpc (P<0.05).7,8-DHF could also protect the DA terminals in the striatum from the toxicity of rotenone compared with the rotenone group. The activity of AEP and the expression level of p-a-synuclein (Ser129)、 p-tau(S396) and tau N368 fragment increased in the substantia nigra and striatum of rotenone group compared with the control group and intervention group (P< 0.05).Compared to the rotenone group, the expression levels of p-TrkB/TrkB and Akt increased in the substantia nigra and the levels of p-MAPK/MAPK, AEP, p-a-synuclein (Ser129), a-synuclein, p-tau(S396) and tau N368 decreased (P<0.05)Conclusions The results revealed that 7,8-DHF could improve the behavioral performance of rotenone-induced PD model and display the neuroprotective effects by the activation of TrkB receptors and its major signaling pathways, the inhibition of abnormal phosphorylation of MAPK, a-synuclein and tau, and the down-regulation of AEP and the produce of tau fragment-cleaved by AEP.