Effects Of α-SynN103/tauN368 Preformed Fibrils Complex On The Axonal Transport Of Rats’ Dopaminergic Neurons And Its Mechanism

Part 1 Effects of Intra-striatal Injection of α-SynN103/Tau N368 Preformed Fibrils Complex on the Structure and Function of Rats’ Substantia Nigra-Striatal Dopamine NeuronsObjection The deposition of LBs and the loss of substantia nigral dopamine neurons are the main pathological features of PD.In PD pathology,LGMN is significantly activated and able to cleave α-Synand tauproteins to form α-SynN103 and tauN368,respectively.The two protein fragments strongly interact with each other and can induce endogenous α-Synaccumulation and formation of LBs.Therefore,α-SynN103/tauN368 preformed fibrils complex(hereafter referred to as PFFs complex)were used to construct an early disease model of PD,to explore the time-dependent changes in α-Synpathology and its pathological effects on nigrostriatal dopamine neurons,and to uncover early pathological events in PD.Methods Male SD rats were intra-striatal injected with PFFs complex(3 μg/μL,10 μL or1.5 μg/μL,10 μL)or an equal volume of PBS prepare an α-Synaggregation model.Behavioral assessments were performed at 1,2,3,and 6 months after completion of intrastriatal injection,and Western blot,immunostaining(including immunofluorescence and immunohistochemistry)and HPLC-ECD experiments were completed at different time points to detect temporal changes in PFFs complex-induced α-Synpathology and nigrostriatal dopamine neurons’ pathological changes.Results The abundance of α-Synpathology in PFFs rats was dose-dependent and persisted throughout the study.The mounts of pathological inclusions peaked at 2 months after intrastriatal injection and caused progressively degenerative changes in substantia nigra-striatal dopamine neurons.After 6 months,the number of nigrostriatal dopamine neurons decreased by 30%.In addition,significant changes in cytoskeleton-associated proteins preceded neuronal death,which was found for the first time in the PFFs model and may be associated with axonal degeneration.Conclusion Intra-striatal injection of PFFs complex constructed a novel model of early stage of PD with extensive temporal correlates of α-Synpathology,axonal degeneration,and nigrostriatal dopamine neuron apoptosis.Notably,cytoskeleton-related protein levels have been altered in this model early in the study,possibly reflecting alterations in microtubules in neuronal axons,and microtubule-based axonal transport functions may have been affected as well.Part 2 Effects of α-SynN103/tauN368 Preformed Fibrils Complex on Axonal Transport Related Proteins in Rats’ Dopaminergic Neurons Objection To clarify the effect of PFFs complex on the expression of dopaminergic neurons’ axonal transport-related proteins after inducing the accumulation of endogenous α-Synphosphorylation in rats’ brain.Methods A rat model with abnormal α-Synaggregation was constructed by intra-striatal injection of PFFs complex(3 μg/μL,5 μL)or an equal volume of PBS.Behavioral assessments were performed 1 and 2 months after completion of striatal injection,and rats with behavioral differences when compared with the PBS controls were selected for subsequent experiments.The expression of kinesin-1 and each component of dynein complex was detected by Western blot in the affected striatum and substantia nigra.The levels of the corresponding m RNAs were assessed through RT-q PCR,and the approximate distribution of kinesin-1 and dynein complex was determined.Given that the two proteins are responsible for the antero-and retrograde axonal transport activities,respectively,we combined the results of Western blot,RT-q PCR and immunofluorescence staining to assess the anterograde transport of mitochondria and NF,and the retrograde transport status of Trk B.Then immunoprecipitation was performed to comprehensively analyze the effect of the PFFs complex on kinesin-1 and dynein based axonal transport.Results PFFs rats showed significantly lower m RNA levels of KHC,KLC1 and KLC2,and much less levels of the three proteins in the striatum compared with PBS controls,but the proteins in the ipsilateral substantia nigra did not change,suggesting that kinesin-1 based anterograde transport may have been impaired and accumulate in the cell body(substantia nigra).In contrast,in the affected striatum,the dynein complex showed an opposite trend to kinesin-1,as the m RNA and protein levels of dynein,dynactin,and snapin were elevated.However,immunoprecipitation results suggested that not only the binding of kinesin-1 to the mitochondrial transport protein MTX2 was reduced and the anterograde of mitochondria was impaired,but also the level of the retrograde transport complex formed by dynein and snapin was decreased,resulting in a weakened interaction of the complex with Trk B.Immunofluorescence staining showed an increased co-localization of Trk B and Rab7 in the affected substantia nigra,further demonstrating that the reverse transport of Trk B may be restricted.In addition,p Erk,which is retrograde transported together with Trk B,also showed intra-synaptic(striatal)accumulation.Conclusion Striatal injection of the PFFs complex significantly reduced the expression level of kinesin and affected the binding of kinesin to mitochondria,resulting in restricted anterograde transport of mitochondria.Despite the upregulation of dynein complex expression levels,p S129 competed with snapin for the binding site of dynein,resulting in reduced interaction of the dynein complex with Trk B,whose reverse transport activity was also restricted.Part 3 Mechanism of Abnormal Axonal Transport in Rats’ Dopaminergic Neurons Caused by Intra-striatal Injection of α-SynN103/tauN368 Preformed Fibrils ComplexObjection To clarify the mechanisms by which PFFs complex caused kinesin-based anterograde axonal dysfunction in rats’ nigrostriatal dopamine neurons,and to explore the ameliorative effects of related inhibitors on transport abnormalities.Methods ⑴ Rats model with abnormal α-Synaggregation were constructed by intrastriatal injection of PFFs complex(3 μg/μL,5 μL)or an equal volume of PBS.Behavioral assessments were performed 1 and 2 months after completion of striatal injection,and PFFstreated rats that showed behavioral differences compared with the PBS controls were selected for subsequent experiments.The expression of PIKE,p-AMPK/AMPK,and p-p38MAPK/p38 MAPK in the affected striatum and substantia nigra was examined using Western blot,and the activation of AMPK downstream pathways was verified by immunofluorescence staining of LC3.⑵ The rats model was constructed using the method mentioned above and randomly divided into PFFs/DMSO group(positive control group),PFFs/AICAR group,PFFs/Compound C group and PBS/DMSO group(negative control group).After 2 months,the lateral ventricle was intervened with the appropriate drug 1 day before sampling.The expression of p-AMPK/AMPK,p-p38 MAPK/p38 MAPK,kinesin-1and mitochondrial transport proteins in the affected striatum was detected by Western blot;the corresponding m RNA levels of the above proteins were also detected by RT-q PCR.⑶On the basis of the establishment of α-Synaberrant aggregation model,the groupings were as follows: PBS/DMSO(negative control group),PFFs/DMSO(positive control group),and PFFs/SB203580 groups,and intraperitoneal injection was performed for 14 consecutive days.Behavioral assessments were performed on day 8 and day 15 of the drug intervention,respectively.The expression of p-p38 MAPK/p38 MAPK,p-MK2/MK2,LC3 II,kinesin-1and mitochondrial transport proteins in the striatum on the diseased side was detected by Western blot;the corresponding m RNA levels of the above proteins were detected by RTq PCR;the immunoprecipitation method was used to assess the binding of KHC to microtubule proteins and KLC1 to mitochondrial transport proteins.Transmission electron microscopy and immunofluorescence staining were used for analysis of structural and functional alterations in dopamine neurons.Results ⑴ The PFFs complex led to a decrease in PIKE expression and induced significant activation of AMPK/p38.⑵ The AMPK activator AICAR further elevated the activation level of AMPK,resulting the expression of kinesin-1 was further reduced,and the anterograde axonal transport of mitochondria was significantly impaired.On the contrary,the AMPK inhibitor inhibited the phosphorylation of AMPK to some extent,which led to a strong rebound in the transcriptional level of kinesin-1 and alleviated the transport restriction of mitochondria.⑶ The p38 MAPK inhibitor SB203580 not only inhibited the activation of p-p38 MAPK on its substrate,but also enhanced the binding of kinesin-1 to microtubules and mitochondria,increased the efficiency of mitochondrial anterotransmission,and improved the structure and function of dopaminergic neurons to some extent.Conclusion The PFFs complex inhibits the expression and function of PIKE by promoting abnormal phosphorylation of endogenous α-Syn,which indirectly activates AMPK and p38 MAPK.The activated AMPK and p38 MAPK impeded the transcription of kinesin-1 and limited the interaction of kinesin-1 with microtubule and mitochondrial transport proteins,respectively,which restricted the kinesin-based anterograde axonal transport process.