Neurotrophic Effect Of CDNF On Dopaminergic Neurons After Proteasomal Inhibition Via Modulating Proteasome Activities

BACKGROUD AND OBJECTIVESParkinson's disease(PD),which affects 1-2%of the population over age 65,is the most common neurodegenerative disordr.The clssical form of the disease is characterized clinically by resting tremor,bardykinesia,rigidity,and postural instability.Its pathologic hallmarks are the selective and progressive loss of dopaminergic neurons of the substantia nigra pars compacta(SNc)and the formation of intraneuronal proteinaceours inclusions known as Lewy bodies within the surviving neurons of the substantia nigra.Although the clinical and pathological phenotype is well-described and clear,the etiology and pathogenesis of PD remain elusive.As far as the therapeutic methods concerned,all the treatments just try to alleviat the symptomatic manifestations,including the drug therapy and operative therapy,which consists of the stereotactic lesion and deep brain stimulation.The clinical signs would be recurrent accompanied with the patholgical aggravation.Thus,it has been considered as the focal research to prevent the progressive DA neurodegeneration in SNc.Neurotrophic factors play a regulatory role in neurogenesis,survival,growth,differentiation and migration,and could be considered as the necessary protein molecules for neuronal growth,survival and functional maintenance.As for the recent studies,the cerebral dopamine neurotrophic factor(CDNF),a member of the mammalian mesencephalic astrocyte-derived neurotrophic factor family,has been shown to significantly protect and rescue the neurodegeneration of DA neurons from 6-OHDA-induced damage and be served as a promising candidate of neurotrophic therapy for PD.6-Hydroxydopamine(6-OHDA),a hydroxylated analogue of dopamine,leaded to the ROS degeneration,which induced the neurodegeneration of DA neurons.However,the dysfunction of proteasome regarded as the responsible pathogenesis of PD.Ups,as a protein degradation pathway in cells,plays an important role in the degradation of intracellular abnormal accumulation proteins,and plays a neuroprotective role through the timely and effective degradation of abnormal intracellular accumulation.Therefore,it is of intrest to evaluate the similar effects of CDNF against DA neurodegeneration induced by UPS inhibitor.The present study aimed to investigate whether CDNF exhibited comparable neuroprotective and reversal effects in proteasomal inhibitor-treated PC 12 cells,and to explore the possible mechanism involved in the 26s proteasomal expression and its multienzymatic proteolytic activities.The present study tried to prove the new assumption of "CDNF-proteasome-protection and reversal of DA neurons",and clarify the proteasomal functional activity induced by CDNF proteins.METHODS The first part:Effect of CDNF on the neurodegeneration of DA neurons induced by proteasomal inhibition.Firstly,the construction of CDNF gene recombinant baculovirus expression vector Bacmid-CDNF transfected Sfs insect cells by Bac-to-Bac protein expression system,according to molecular cloning method to complete the purification of CDNF protein expression,and identification,to prepare for the follow-up experiment.The PC 12 cells were induced to be PD models by proteasomal inhibitors.Five groups were designed,as control group,MG132 group,Lactacystin group,CDNF+MG132 group and CDNF+Lactacystin group.PC 12 cells were maintained in 10%heat-inactivated newborn calf serum and 100 U/ml penicillin/streptomycin.The cells were seeded at a density of 2×105 cells/ml in 96-well plates and incubated for 24 h.To investigate the protective effect of CDNF,the cells were preincubated with 200 nM CDNF protein for 6 h,and exposed to 12.5?M lactacystin or 5?M MG 132,respectively,for 24 h.To investigate the reversible effect of CDNF,PC 12 cells were pre-treated with 12.5?M Lactacystin or 5?M MG132 for 24 h,and incubated with the same doses of CDNF protein for 24 h.All experiments were replicated three times.Finally,the PC 12 cells activities were measured by MTT assay,cells morphologies were detected by TH immunofluorescenee assay and the expression of alpha-synuclein protein were detected by immunofluorescence staining.The second part:Regulations of proteasomal expression and its hydrolase activities after proteasomal inhibition by CDNF.Firstly,the construction of CDNF gene recombinant baculovirus expression vector Bacmid-CDNF transfected Sf9 insect cells by Bac-to-Bac protein expression system,according to molecular cloning method to complete the purification of CDNF protein expression,and identification,to prepare for the follow-up experiment.The PC 12 cells were induced to be PD models by proteasomal inhibitors.Five groups were designed,as control group,MG132 group,Lactacystin group,CDNF+MG132 group and CDNF+Lactacystin group.PC 12 cells were maintained in 10%heat-inactivated newborn calf serum and 100 U/ml penicillin/streptomycin.The cells were seeded at a density of.2×105 cells/ml in 96-well plates and incubated for 24 h.To investigate the protective effect of CDNF,the cells were preincubated with 200 nM CDNF protein for 6 h,and exposed to 12.5?M lactacystin or 5?M MG132,respectively,for 24 h.To investigate the reversible effect of CDNF,PC12 cells were pre-treated with 12.5?M Lactacystin or 5?M MG132 for 24 h,and incubated with the same doses of CDNF protein for 24 h.Finally,the expression of 26S proteasome and the contents of glutamyl hydrolase,chymotrypsin and trypsin were detected by ELISA assay.The expressions of proteasome and three kinds of enzymes by Lactacystin/MG132 administration after treated by CDNF were analyzed statistically.RESULTS The first part:Effect of CDNF on the neurodegeneration of DA neurons induced by proteasomal inhibition.As far as the cell activities concerned,after exposure to 12.5?M Lactacystin or 5?M MG 132 for 24 h,the cells viabilities decreased ratios of PC 12 cells were detected as 79.1%and 78.6%respectively by MTT assay,no statistical significance between the two goups.Morphological analysis of TH-immunofluorescence staining demonstrated nuclear condensation and fragmentation in the Lactacystin/MG132 groups,both of which are typical hallmarks of apoptosis.To investigate the protective effect of CDNF,the cells were preincubated with 200 nM CDNF protein for 6 h,and exposed to 12.5?M lactacystin or 5?M MG 132 for 24 h.The cells viabilities decreased ratios of PC12 cells were down-regulated to 52.3%and 49.7%respectively,with statistical significance between the Lactacystin/MG132 groups and CDNF+Lactacystin/MG132.The number of cells also increased compared with non-treated groups.To investigate the reversible effect of CDNF,the cell were first exposed to 12.5?M lactacystin or 5?M MG 132 for 24 h,and incubated with 200 nM CDNF protein for 24 h.The cells viabilities decreased ratios of PC12 cells were down-regulated to 59.5%and 56.2%respectively,respectively,with statistical significance between the Lactacystin/MG132 groups and Lactacystin/MG 132+ CDNF.The number of cells also increased compared with non-treated groups.Compared with the control panel,alpha-synuclein immunofluorescence was observed in a number of lactacystin-and MGI32-treated PC 12 cells with high expression of fluorescence,the fluorescence intensity in lactacystin-and MG132-treated PC 12 cell were quantified as 2.1755 and 2.8990 respectively.When pre-treated with 200 nM CDNF protein for 6 h,the number of PC 12 cells with immunofluorescence was reduced,and the fluorescence intensity faded,while the fluorescence intensity in lactacystin-and MG132-treated PC 12 cells decreased to 1.3871 and 1.1710 respectively,with statistical significance between the Lactacystin/MG 132 groups and CDNF+ Lactacystin/MG132.With respect to the samples post-treated with CDNF,the number of cells and fluorescence intensity also decreased,and the fluorescence intensity in lactacystin-and MG132-treated PC 12 cells decreased to 1.4899 and 1.2958 respectively,with statistical significance between the Lactacystin/MG 132 groups and Lactacystin/MG 132+CDNF.The second part:Regulations of proteasomal expression and its hydrolase activities after proteasomal inhibition by CDNF.As far as the cell activities conceerned,after exposure to 12.5?M Lactacystin or 5?M MG132 for 24 h,the activity of 26s proteasome significantly decreased compared with the no-treated groups.To investigate the protective effect on 26s proteasome by CDNF:when pre-treated with CDNF,the upregulation of 26s proteasome activity was observed in the lactacystin and MG 132 groups,with a greater effect in the latter MG132 group.To investigate the reversal effect on 26s proteasome by CDNF:when post-treated with CDNF protein,upregulation occurred in the MG132 group,but not in the lactacystin group.As for the hydrolases activities in the 20s proteasome.After exposure to lactacystin and MG132,the activities of glutamyl peptide hydrolase,chymotrypsin,and trypsin were significantly decreased(p<0.05).When pre-treated with CDNF,upregulations of glutamyl peptide hydrolase,chymotrypsin and trypsin were observed in the MG 132 group(p<0.05),while only glutamyl peptide hydrolase expression was increased in the lactacystin group(p<0.05).Finally,in the group pre-treated with lactacystin and MG132,only glutamyl peptide hydrolase was reversibly up-regulated compared with the other enzymes(Fig.4D-F).CONCLUSION1.CDNF plays the protective and reversing effect on PC 12 degeneration administrated with MG132/Lactacystin,and without statistical significance between the two groups.2.CDNF plays the protective effect on 26S proteasome induced by MG132/Lactacystin,and CDNF+MG132 group better than the CDNF+Lactacystin group.The reversal effect on 26s proteasome has identified in the MG132+CDNF group,while no statistical reversal effect was detected in the Lactacystin+CDNF group.3.CDNF plays a protective effects on glutamyl hydrolase induced by MG132/Lacatacystin,without statistical difference between the two groups.And,CDNF plays a reverse effect on glutamyl hydrolase induced by MG 13 2/Lacatacy stin,and MG 13 2+CDNF group is better than the Lactacystin+CDNF group.4.CDNF plays a protective effect on chymotrypsin induced by MG 132,with no protective effect observed in the CDNF+Lactacystin group,Lactacystin+CDNF group and MG132+CDNF group,although MG132+CDNF better than the Lactacystin+CDNF.5.CDNF plays a protective effect on Trypsin induced by MG132,with no protective effect observed in the CDNF+Lactacystin group,Lactacystin+CDNF group and MG132+CDNF group.