Parkin. And Interleukin-1¦Â In Parkinson's Disease Research

PartⅠMutations in the Parkin gene are associated with autosomal recessive juvenile parkinsonism(AR-JP),accounting for more than 50%of early onset PD cases.Parkin, as an ubiquitin E3 ligase,provides an important link between protein ubiquitination and the ubiquitin-proteasome system(UPS) in the pathogenesis of PD.A number of Parkin substrates have been identified,including CDCrel-1,CDCrel-2a,synphilin-1, glycosylatedα-synuclein,Pael-R,misfolded DJ-1,Hsp70,etc.Several recent papers have shown that expression of parkin is beneficial to dopaminergic neurons as it protects them from a number of damaging events such as overexpression of mutant forms ofα-synuclein,Pael-R,CDCrel-1,misfolded DJ-1 or neurotoxin MPTP and 6-OHDA.Parkin is also reported to be protective against ER stress,proteasomal inhibition,kainic acid 'excitotoxicity' and ceramide-induced mitochondrial apoptosis. However,Parkin deficient mice display little or no alteration in dopamine neuron survival,but show reduced mitochondrial function.To determine the protective effect of parkin in PD process in vivo,we generated Parkin transgenic mice in which the expression of parkin is confined to neurons.We examined whether MPTP induced less neurotoxicity in Parkin transgenic mice,the results are summarized as follows:1.Transgenic mice showed transgene expression at mRNA and protein levels.Offsprings of different founder mice were screened for transgene expression at both mRNA and protein levels,we finally got two transgenic mouse lines.The transgenic mice showed exogenous Parkin transcription and elevated Parkin protein expression in the cortex,hippocampus,striatum and substantia nigra.Creysl violet staining of different brain regions revealed no change between transgenic mice and wild type littermates.2.Young and old Parkin transgenic mice displayed different protection patterns of dopamine neurons after MPTP administration.We examined TH protein expression in the striatum and TH positive neurons in the substantia nigra after MPTP administration.Compared to wild type litermates,young Parkin transgenic mice showed less reduction of TH protein expression in the striatum 3 days after MPTP toxication,while TH positive cells in the SN were less decreased 1day after MPTP treatment.MPTP induced less neurotoxicity both at 1 day and 3 days after MPTP administration in old transgenic mice.These data indicate that Parkin protects DA neurons from MPTP toxicity.3.DA and its metabolites didn't show significant change in young Parkin transgenic mice and wild type littermates.Measured by HPLC,striatal DA and its metabolites showed decreased concentrations after MPTP treatment.No significant change was observed between Parkin transgenic mice and wild type littermates.It suggests Parkin may be not involved in DA metabolism.4.MPTP elicited less mitochondrial impairment in Parkin transgenic mice.MPTP is reported to impair mitochondrial function.In order to examine whether Parkin could protect mitochondria,we observed morphology of mitochondria in the substantia nigra pars compacta after MPTP administration.Electron microscopy showed increased abnormal mitochondria with numerous vacuoles and fragmented cristae in wild type mice after MPTP treatment,while less impaired mitochondria were found in Parkin transgenic mice.This indicates that Parkin can protect mitochondria from MPTP toxicity.5.Transcriptional expression of bcl-2,bax,iNOS and DJ-1 altered within groups.We then examined the transcriptional expression of mitochondrial related genes, such as bcl-2,bax,DJ-1 and PINK1,pro-inflammation molecules iNOS and TNFα, and other related genes such as CHIP and UCH-L1.We found that bcl-2 and DJ-1 remained up-regulated in the young Parkin transgenic mice,while the up-regulation of bcl-2 and down-regulation of bax and CHIP were detected in the old Parkin transgenic mice 3 days after MPTP administration.These data may in part explain the beneficial role of Parkin.6.α-synuclein protein expression in the striatum was reduced in old Parkin transgenic mice,while Hsp70 expression remained unchanged.As one of the substrate proteins of Parkin,Hsp70 can inhibit mitochondrial apoptosis.Thus,we detected the protein expression of Hsp70 in the striatum within groups and revealed no significant change,α-synuclein,which is involved in the formation of Lewy Body in PD,showed decreased protein expression in old Parkin transgenic mice.This may in part explain that Parkin protects DA neurons through down regulatingα-synuclein to reduce protein aggregation during PD process. Conclusion:1.Parkin attenuates dopaminergic neurodegeneration induced by MPTP through protection of mitochondria.2.MPTP-induced depletion of the striatal DA and its metabolites is not protected by Parkin.3.Downregulation ofα-synuclein protein expression in the old Parkin transgenic mice may in part explain the protection of Parkin. PartⅡThere is increasing evidence that recognition of the possible role of neuroinflammation is a causative factor in the pathogenesis of PD.Glial reaction, which is featured by activation of microglia and astrocytes,is observed in brain tissues of postmortem PD patients and MPTP-induced animal models.IL-1β,an inflammatory cytokine released mainly from glial cells,plays important roles in mediating cellular responses to injuries in the central nerve system.The expression of IL-1βhas been reported to be elevated in the striatum and substantia nigra of PD patients,suggesting its association with PD.However,findings about the role of IL-1βduring brain damage were contradictoryreported.Moreover,greater prevalence of PD in men compared to women is reported.Estrogen is conceived reported to exert dopaminergic neuroprotection both in human and PD mice model,this may be one of the explanations of the gender difference in PD.To determine the changes of IL-1βexpression and the association between the IL-1βexpression and the prevalence of PD in aged mice.By using a sensitive transgenic mice model in which the expression of luciferase reporter gene is under the control of human IL-1β.gene promoter,we examined the IL-1βgene expression pattern in vivo after subacute MPTP toxication in old male and female mice.The results are summarized as follows:1.Old male mice showed dramatically elevated luciferase signals in a flexuous manner at early period of time,meanwhile,the changes of luciferase signals in female were more moderate.Old male and female mice were screened for luciferase expression at different time points during and after MPTP or saline administration.The luciferase activity increased in both MPTP groups.By 49h,the enzyme activity in female mice treated with MPTP had returned back to baseline,while the activity in male mice still sustained at a higher level.Data quantification showed different patterns of MPTP-induced luciferase signals between male and female.Though both sexes revealed significant higher levels of luciferase expression,the changes in male mice were especially vigorous,but quite moderate in female.2.MPTP elicited less dopaminergic toxicity in old female mice than male ones.65h after MPTP administration,we observed a significant depletion of striatal TH protein expression in both sexes,however,TH reduction was significantly greater in old male mice when compared to female.The TH positive neurons in the substantia nigra of old female mice didn't show prominent difference between the saline and MPTP group,while significant decrease of TH positive cells was seen in male MPTP intoxicated mice3.The transcriptions of some inflammatory and apoptotic molecules in the substantia nigra showed no significant change between groups.65h after the last MPTP injection,transcriptional levels of cytokine TNF-αand IL-6,pro-inflammatory molecule iNOS,together with apoptosis related molecules bcl-2 and bax in the substantia nigra were detected by real-time PCR.The expression of these genes showed no significant alteration between groups at this time point, although IL-6 gene trancription was increased after MPTP administration,the p-value of IL-6 between the control and MPTP group in female mice was 0.07.4.MPTP intoxication increased GFAP protein expression,but didn't change BDNF expression in the striatum of both old male and female mice.GFAP and BDNF protein expression in the striatum was were measured through western blot analysis.MPTP intoxication induced robust increase of GFAP expression in both sexes 65h after the last MPTP injection.Only slightly higher expression of BDNF was observed in old female mice,however,no significant difference was observed obtained between groups.Conclusion:1.Elevated and dramatic changes of IL-1βgene expression in old male mice at early period of time may be responsible for the DA neuron susceptibility to MPTP in old male mice.2.MPTP elicited less dopaminergic toxicity in old female than male mice.