Establishment Of Chronic And Progressive Parkinson’s Disease In Rhesus Monkeys And Neuroprotective Effect And Mechanism Of Iron Chelator Clioquinol On PD

Parkinson’s disease,as the second largest neurodegeneration disorder,its clinical diagnosis is mainly depending on the classic motor deficits which will be presented only after 70%-80%dopamine(DA)neurons in the substantia nigra(SN)are lost,and there are no any biomarkers can be used for early diagnosis at present.At present,levodopa(LD),the standard therapy drug,can just supply the body with additional amount of dopamine,but can’t offer any help to the improvement of pathology,meanwhile it will cause serious side effects after 5-10 years of use.So,it’s very urgent to figure out the specific pathogenesis of PD and related new drugs for PD treatment,as well as biomarkers for early diagnosis.And animal models are the best platforms for biomarkers,pathogenesis and new drugs study.Among the PD animal models,non-human primate model induced by MPTP is the best.But some defects,such as unstabled and easily recovered clinical phenotypes in the acute or sub-chronic model,varied degrees of clinical phenotypes of the model even if accepted the same dose of MPTP,cannot imitate resting tremor and lewy bodies,of the models are still unsolved at present.Therefore,an ideal non-human primate PD model that can imitate almost all the clinical phenotypes of PD,will play an important and irreplaceable role in the in-depth study of PD pathogenesis,the screening of risk factors for PD early-diagnosis,and the evaluation of new drugs in the preclinical period.The etiology of PD is complex,related to gene mutation,aging,environmental toxin,oxidative stress,and neuroinflammation.Excess Iron induced oxidative damage plays an important role in the pathogenesis of PD,but the specific relationship between iron and PD,especially in the early stage(before the appearance of typical clinical symptoms)of PD,is not very clear.Therefore,the present studies aim to establish an ideal non-human primate model that can simulate almost all the clinical phenotypes of PD,monitor the progress of the model by testing behavioral scores and candidate genes expression during the overall periods,in-depth study the relationship between iron and PD in both early and advanced stages,and remove the excess iron in the SN by using iron chelator clioquinol and evaluate the therapeutic effect and study the possible therapeutic mechanisms,expecting to provide evidence and support for the clinical application of iron chelators in neurodegeneration diseases.Exp.1 Establishment of PD model in aging rhesus monkeys and analysis of relationship between iron and PDTotally,six aging monkeys were randomly divided into two groups:MPTP group and normal group.MPTP group monkeys were injected with a small dose(0.2 mg/kg)of MPTP for 45 days.Specifically,there were two periods of MPTP injection,the first lasting for 15 days(1st MPTP)and the second for 30 days(2nd MPTP).Both MPTP-injection periods were followed by an interval of withdraw period,8 and 7weeks,respectively.Normal group were administered with same dose of saline.Here,we reported a significant iron elevated accompanied by DA degeneration in the SN in Mk3 that displayed the most severe PD behavioral deficits.The other two monkeys in MPTP group displayed less severe PD motor deficits and pathologies,however,their SN iron levels in the SN were significantly lower than controls.Similarly,the results of in vitro system showed that high doses of ferric ammonium citrate(FAC),a factor known to enhance iron accumulation,increased MPP~+-induced cell death in both U251 and SH-SY5Y cells,and even in control cells.However,low dose of FAC restored or increased the viability of U251 and SH-SY5Y cells in the absence or presence of MPP~+.These findings suggest that high iron may indicate and contribute to heightened MPTP/MPP~+-induced PD pathology in late stage of PD,while depressed level of iron may offer neuroprotective in early stage of PD.Exp.2 Establishment of PD model in young rhesus monkeys and analysis of relationship between serum iron content,candidate genes expression and PD progressTotally,twelve young monkeys,three for control group and nine for MPTP group,were used.The MPTP injection procedure was updated timely according to the behavior test result,simply,at the beginning of the experiment,the MPTP dose of all monkeys was 0.2mg/kg,and at the end of experiment,the dose of some monkeys was increased to 0.5mg/kg.During the overall procedure,serum iron contents tested by blood chemistry,candidate genes expressions in the blood tested by RT-q PCR,and the progress of PD elevated by behavior scores,were analyzed along with the protocol.Meanwhile,at the end of the experiment,MRI and EMG tests were used to evaluate the model.Both MRI and EMG results indicated that the monkey model was very successful.Behavior test result showed that all the MPTP group monkeys showed mild PD symptoms since the 9th week and gradually reached a classic and stable Parkinsonism stage at the 18th week.Meanwhile,MPTP group monkeys were divided into 3 sub-groups,the sensitive group firstly presented with motor deficits,the mild-sensitive group secondly presented with motor deficits,and the non-sensitive group lastly presented with motor deficits.Interestingly,the serum iron content in sensitive group was first significantly increased,and that of in non-sensitive group was the last.So the serum iron contents were closely related to the development of PD.The RT-q PCR results indicated that the genes of UCHL1,PARKIN,USP30,MUL1 and PINK1 m RNA in blood significantly changed at early stages,may act as early biomarkers,and LRRK2,TRIM24,and DJ-1 m RNA in blood significantly changed at late stages,may act as late biomarkers.Therefore these genes expressed in blood can be act as biomarkers to monitor the progress of PD,and may also facilitate the early diagnosis and treatment prognosis.Exp.3 Evaluation of the therapeutic effect of iron chelator CQ on PD monkey model and the exploration of CQ therapeutic mechanismsTotally,twelve young macaques(three for control group and nine for MPTP group)were used,and the MPTP injection protocol was the same with experiment 2.When the classical clinical behavior phenotypes were stabled for 3 weeks after the withdraw of MPTP,MPTP group monkeys were randomly divided into three sub-groups,MPTP group,MPTP+LD group and MPTP+CQ group,and which were accepted with levodopa(LD,12 mg/kg)twice daily,CQ(10 mg/kg)twice daily,and solvent twice daily,for 4 weeks,respectively.Behavioral results showed that the motor deficits in both MPTP+LD and MPTP+CQ animals were significantly improved,when compared to MPTP group.Serum iron content in the blood of MPTP+CQ was in the normal level and the candidate genes expression was also reversed in a certain degree when compared to MPTP group.Histopathological results showed that DA neurons in the SN of MPTP+LD and MPTP+CQ groups were significantly increased compared to MPTP group.The above results indicate that CQ can play a protective role in PD.Therefore,we explored the therapeutic mechanism of CQ on PD.Results showed that iron content,ROS levels and neuroinflammation levels were decreased to normal level after the treatment of CQ when compared to MPTP group,and damaged astrocytes were also repaired to normal level.In addition,the in vivo results showed that CQ could increase the cell viability,inhibit ROS levels and suppress neuroinflammation after MPP~+injured in U251 cells,but have no any protective effect on SH-SY5Y cells.However,the U251 cell culture media pretreated with CQ could significantly increase the cell viability after MPP~+injured in SH-SY5Y cells when compared to that of U251 cell culture media without CQ treatment.Therefore,the above results indicate that on one hand,CQ could specifically remove the excess iron in the SN,weaken the oxidative stress and suppress neuroinflammation,on the other hand CQ could repair the damaged astrocytes by regulating the AKT-m TOR pathway to against apoptosis and to promote autophagy,therefore secreting glial-derived neurotrophic factors to protect DA neurons.In summary,the PD monkey model in the present experiments replicated almost all the motor deficits,including typical motor deficits(resting/postural tremor,bradykinesia,freezing),and non-motor deficits,including lethargy,constipation,salivation,mask face,low-reactivity socialization,etc.,which could be an excellent model benefit for PD pathological mechanisms and new drug study.Meanwhile,iron levels in the blood and in the SN were closely related to the pathogenesis of PD,clioquinol,as an iron chelator,on one hand can specifically remove the excess iron in the SN and attenuate oxidative stress and neuroinflammation,on the other hand it can repair the damaged astrocytes and then protect DA neurons through AKT-m TOR pathway.