AEP Cleaves SYNJ1 And Regulates The Pathogenesis In Parkinson's Disease

BackgroundParkinson's disease(PD)is one of the most common neurodegenerative diseases,which is characterized by the loss of dopaminergic neurons in the nigrostriatal pathway.Most studies focus on the deformation and loss of dopaminergic neurons.Recently,more studies have identified other pathological changes before neuronal loss,such as axon degeneration and synaptic dysfunction.Moreover,synaptic dysfunction can impair the loading of dopamine in neurons and more importantly,the dopaminergic neurons are more prone to degenerate compared to other neurons in the brain.However,the molecular mechanisms of synaptic dysfunction and dopaminergic neuronal susceptibility in PD is still unclear.Exploring the mechanisms of synaptic dysfunction in the pathogenesis of PD and the susceptibility of dopaminergic neurons can facilitate the early intervention for PD.Asparagine endopeptidase(AEP)is a lysosomal cysteine protease that cleaves the protein substrate on the C-terminal side of asparagine.Previous reports have proved that neuronal AEP is activated during aging,which is the main risk factor for PD.Previous studies have found that AEP activity in the substantia nigra increases in an age-induced manner,and it cuts?-synuclein(?-syn)and generates?-syn fragments that are prone to aggregate.Deleting of AEP from the PD mouse model eliminated the damage of dopaminergic neurons.These suggest that the abnormal activation of AEP in the brain is involved in the occurrence of PD.Synaptojanin 1(SYNJ1)is a phosphatidylinositol phosphatase that is expressed at a high level in neurons and is enriched in synaptic structures.SYNJ1 participates in the physiological process of synaptic vesicle endocytosis and its circulation in the presynaptic membrane through its phosphatidyl muscle on the alcohol phosphatase activity of SYNJ1protein and the interaction with endocytosis-related proteins,and participates in the synapse metabolic processes of various phosphatidylinositols in the presynaptic membrane.More importantly,the SYNJ1 mutant causes recessive genetic early-onset Parkinson's disease,so it was named PARK20.Related studies have found that SYNJ1mutants cause neuronal synaptic vesicle endocytosis(SVE)dysfunction and early endocytic corpuscle formation disorder,and induce PD-like pathological manifestations.However,in sporadic PD,the changes of SYNJ1 and its pathogenic mechanism are still elusive.We suspect that in sporadic PD,AEP is abnormally activated with age and cleaves the SYNJ1 protein,which leads to the variability of dopaminergic neurons.The present study uses AEP gene knockout(AEP^-/-)mice and human?-syn A53T transgenic(A53T)mice to explore the shearing effect of AEP on SYNJ1 protein and its pathogenesis in PD.MethodPart one:The brain tissues of wild-type(WT)mice and AEP^-/-mice were send to mass spectrometry to analyze the SYNJ1 fragments.We generated a plasmid encoding full-length SYNJ1 protein with a Flag-his tag at the N-terminal or a full-length SYNJ1protein with a HA-tag at the C-terminal.After transfection,the SYNJ1 full-length protein and the activated recombinant AEP are incubated together,and analyzed using Western blot(WB).We also constructed the N599A SYNJ1 point mutation plasmids,and performed AEP in vitro digestion assay.We generated SYNJ1 N599 antibody,and confirmed the specificity of anti-N599 antibody by WB and immunohistochemistry(IHC)experiment.Human brain samples from PD patients and control groups of the same age were used to complete WB and IHC experiments to detect SYNJ1 fragments.Brain slices from WT,A53T and AEP^-/-mice over 12 months old were used to complete IHC experiments to detect N599 fragments.Part two:Purified Flag-his-SYNJ1 full-length and fragments were used for phosphatase activity detection.GST pull-down assay were used to detect the interaction of purified full-length and fragmented SYNJ1 with endocytosis-related proteins.The full-length and fragmented SYNJ1 were overexpressed in primary cultured neurons,and immunofluorescence,FM 4-64 endocytosis experiments,Dil staining,membrane electrophysiological experiments and apoptosis experiments were performed.Transferrin reuptake experiment were performed in COS-7 cells expressing full-length and fragmented SYNJ1.The full-length and fragments of SYNJ1 were overexpressed in the A53T hippocampus to test their impact on the electrophysiological properties of neurons.Part three:AAVs encoding SYNJ1 full-length,fragments and N599A point mutation were injected into the substantia nigra of 2-month-old A53T mice.The injection site was tested 4 weeks after the injection.Behavioral experiment was completed 16weeks after the injection,including the hindlimb clasping test,rotarod test,the cylinder test,balance beam test,grip test and footprint test,as well as tyrosine hydroxylase(TH)and dopamine transporter(DAT)immunohistochemistry experiments.Results1.AEP cleaves SYNJ1 during the pathogenesis of PDMass spectrometry analysis confirmed that compared with AEP^-/-mice,SYNJ1fragments in the brain tissue of WT mice were significantly increased(P<0.05).WB experiments confirmed that the full-length of SYNJ1 was cut into two fragments by AEP:fragments 1-599 containing the N-terminus and fragment 600-1309 containing the C-terminus.The AEP digestion experiment of point mutation SYNJ1 confirmed that SYNJ1was cut at the N599 site of SYNJ1.In the WB experiment of human brain samples,compared with the control group,the full-length SYNJ1(SYNJ1 FL)in PD patients was significantly reduced,while the fragment of N599 in the human brain sample IHC experiment was significantly increased compared with the control group(P<0.05).IHC experiments shown that compared with WT and AEP^-/-mice,the signals of fragments 1-599 in the brain slices of A53T mice were significantly higher(P<0.05).2.AEP-generated SYNJ1 fragment induces synaptic dysfunction and neuronal degeneration.The purified full-length and fragments of SYNJ1 were reacted with PI(3)P,PI(4,5)P₂,PI(3,4,5)P₃ substrates and the phosphatase activity was detected.The results shown that when the substrate was PI(4,5)P₂,PI(3,4,5)P₃,the phosphatase activity of SYNJ1fragment was significantly reduced(P<0.05).When the substrate was PI(3)P,SYNJ1(1-599)fragment,compared with SYNJ1(600-1309)fragment,it still had phosphatase activity(P<0.05).The results of the pull-down experiment shown that SYNJ1 FL interacted with Endophilin A1(Endo A1),Amphiphysin I/II(AMPH I/II),and Dynamin I;while SYNJ1(600-1309)fragment did not interact with Dynamin I,but also interacted with other proteins.The SYNJ1(1-599)fragment did not interact with the above endocytic proteins.In COS-7 cells,the transferrin uptake in the SYNJ1(1-599)and SYNJ1(600-1309)groups was significantly reduced(P<0.05)when compared with the SYNJ1 FL group.After overexpressing SYNJ1 FL,SYNJ1(1-599)and SYNJ1(600-1309)in primary cultured neurons,immunofluorescence was used to detect the co-localization of AMPH I,Endo A1,Dynamin I and overexpressed proteins in neurons.The results confirmed that SYNJ1(1-599)did not co-localized with endocytic proteins,while AMPH I and Endo A1 co-localized with SYNJ1(600-1309).In the FM 4-64 endocytosis experiment,compared with the SYNJ1 FL group,the FM 4-64 endocytosis rate of the SYNJ1(1-599)and SYNJ1(600-1309)groups was significantly reduced(P<0.05).Immunofluorescence shown that the endocytosis marker early endosome antigen 1(EEA1)in the SYNJ1(1-599)group was significantly reduced compared to other groups(P<0.05),while the fluorescence cluster density of the Golgi marker was similar in the three groups(P>0.05).The results of patch clamp electrophysiological experiments shown that compared with neurons overexpressing SYNJ1 FL and SYNJ1(600-1309),the spontaneous excitatory postsynaptic currents(s EPSCs)of neurons overexpressing SYNJ1(1-599)was impaired.The frequency was significantly reduced(P<0.05),and the average amplitude did not change significantly(P>0.05).Compared with the control group,the frequency and average amplitude of spontaneous inhibitory postsynaptic currents(s IPSCs)did not change significantly(P>0.05).Similarly,SYNJ1(1-599)dendritic spines were significantly reduced(P<0.05).The results of apoptotic cell detection shown that SYNJ1(1-599)compared with SYNJ1 FL and SYNJ1(600-1309)groups had more apoptotic cells(P<0.05).SYNJ1 FL,SYNJ1(1-599)and SYNJ1(600-1309)were overexpressed in the hippocampus of mice.The results of brain slice electrophysiological experiments showed that compared with other groups,the frequency and amplitude of miniature postsynaptic currents(m EPSCs)in hippocampal slices of SYNJ1(1-599)group were significantly reduced(P<0.05).Compared with the control group and the SYNJ1 FL group,the paired-pulse facilitation ratio of the SYNJ1(1-599)and SYNJ1(600-1309)groups was significantly reduced(P<0.05).The immunofluorescence results of brain slices shown that,compared with the SYNJ1 FL group,the substantia nigra synaptic vesicle markers were reduced in mice overexpressing the fragments of SYNJ1(P<0.05).3.AEP-mediated cleavage of SYNJ1 promotes the pathogenesis of PD.SYNJ1 FL,SYNJ1(1-599),SYNJ1(600-1309)and SYNJ1 N599A were overexpressed in substantia nigra of A53T mice.The results of immunohistochemistry shown that compared with the control group,SYNJ1 FL and SYNJ1(600-1309),TH-positive cells and the IHC optical density of DAT in the SYNJ1(1-599)group was significantly reduced(P<0.05).Compared with SYNJ1 FL,in the SYNJ1 N599A group,the TH-positive cells and IHC optical density of DAT increased(P<0.05).In the results of behavioral experiments,the SYNJ1(1-599)group had obvious dyskinesias and incoordination,compared with the vector,SYNJ1 FL and SYNJ1(600-1309)group(P<0.05).Compared with SYNJ1 FL,SYNJ1 N599A group did not show dyskinesia in some behavior tests(P<0.05).Conclusions1.SYNJ1 is a physiological substrate of AEP,which is activated during the pathogenesis of PD and cleaves SYNJ1 to generate the SYNJ1(1-599)and SYNJ1(600-1309)fragments.3.AEP-generated SYNJ1(1-599)fragment leads to synaptic dysfunction and degeneration of dopaminergic neurons in A53T mice,and promotes motor deficits in SNCA A53T mice.7.Blocking the cleavage of SYNJ1 by AEP reduces neuronal defects and motor impairments in SNCA A53T mice.