Dysfunction Of Glymphatic System And Accumulation Of Alpha-synuclein Induced Parkinson’s Disease-like Changes

Parkinson’s disease（PD）is the second-largest neurodegenerative disease in the world.Its core pathological mechanism is the generation,aggregation and"prion"-like transmission of the pathogenic form ofα-synuclein（αSyn）（oligomers,mutants,phosphorylation,etc.）.αSyn is not only the main component of Lewy bodies（LBs）but also one of the main causes of extrapyramidal dyskinesia（resting tremor,retardation,muscle rigidity and postural and gait disorders）and non-motor symptoms（such as cognition,gastrointestinal tract,pain,sleep and Urinary dysfunction,etc.）.To delay the progression of the disease,the elimination of targeted pathogenicαSyn has become an important strategy for PD disease modification treatment.Studies have shown that the glymphatic system plays a key role in the clearance ofβ-amyloid（Aβ）and other central macromolecular substances.Preliminary laboratory studies also found that the glymphatic system is involved in the clearance of centralαSyn,suggesting that glymphatic pathway dysfunction may be related to the occurrence and development of PD.On this basis,the present study further explored the relationship between the impairment of the glymphatic system and the accumulation ofαSyn and observed the effects on PD-like manifestations.To investigate the relationship between overexpression ofαSyn and glymphatic system disorders,we first explore the relationship between glymphatic pathway dysfunction andαSyn clearance.First,the effects of the glymphatic system on the accumulation of pathologicαSyn,the loss of dopaminergic neurons,and PD-related behavior were investigated using a model of glymphatic system dysfunction mediated by Aquaporin 4（AQP4）knockout.Subsequently,by constructing transgenic mice with overexpressing human-derivedαSyn in the whole brain neurons,the effect ofαSyn overexpression on the expression and localization of AQP4 was explored.Finally,based on overexpression ofαSyn,the function of the glymphatic system was further interfered with to investigate the effect of accumulation ofαSyn and glymphatic system disorders on the pathologic phenotypes associated with PD.In this study,we found that the impaired clearance of the glymphatic system caused by AQP4 deletion aggravated the accumulation of pathologicalαSyn and PD-related pathological manifestations.Overexpression ofαSyn would also affected the efficiency of the glymphatic system.It is suggested that the accumulation ofαSyn and the glymphatic system interacts with each other and is closely related to the occurrence and development of PD.The intervention of either of them in the early stage of the disease may delay or block the disease’s course,providing a beneficial idea for the targeted therapy of PD.AIM:To investigate the influence ofαSyn accumulation on the glymphatic system and Parkinson’s disease-like manifestations.METHODS:（1）Using AQP4 KO mice as a glymphatic pathway dysfunction model,the fluorescent tracers A488-ca and Evans Blue（EB）were injected into the cerebellar cisternus to detect the degree of penetration into the brain parenchyma;（2）Evens Blue（EB）was injected through the tail vein of mice to observe the extent of EB penetration through BBB and to detect BBB integrity;The effects of AQP4knockout on lateral ventricle and whole-brain neurons were observed by Nissl staining.（3）Immunofluorescence technique was used to observe the influence of glymphatic system disorders on the expression and distribution of Tyrosine hydroxylase（TH）,Neurofilament（NF）andαSyn.（4）Application of Food Burying Test,Rotarod Test,Pole Test and Open Field Test to characterize AQP4 mice PD-related behavior.（5）Observation of glymphatic pathway dysfunction on the pathological phosphorylation ofαSyn and astrocytes marker glial fibrillary acidic protein（GFAP）and TH expression in the main brain by western blotting（WB）technology;application of immunofluorescence technology to detect localization and morphology of dopaminergic neurons and phosphorylatedαSyn in the substantia nigra compact part of the midbrain.（6）Application of visible light imaging technology,gene identification technology,WB and immunofluorescence technology to detect humanαSyn expression in the whole brain of h SNCA^fl/fl;Nestin^Cretransgenic mice;（7）Application of Rotarod Test,Pole Test and Open Field Test to characterize h SNCA^fl/fl;Nestin^Cremice PD-related behavior.（8）Application of WB and immunofluorescence technology to detect AQP4 expression and polarity distribution in various brain regions of A53T mice and h SNCA^fl/fl;Nestin^Cremice;（9）Preparation of glymphatic pathway dysfunction model-acetazolamid（AZA）intraperitoneal injection model,using WB technology to detect midbrain and striatumαSyn and phosphorylatedαSyn expression;Postsynaptic density 95（PSD95）and synaptophysin-38（SYP38）was detected in the midbrain and striatum.RESULTS:（1）Using the fluorescent tracers A488-ca and EB to observe glymphatic transport,AQP4 KO mice showed that the degree of infiltration of into the brain parenchyma within 30 minutes was lower than that of wild-type mice,indicating that the glymphatic system is affected;（2）Using EB to observe the integrity of BBB,AQP4 KO mice showed that EB concentration in the brain of WT and KO mice was very low and showed no difference,suggesting that AQP4 deficiency had no effect on BBB integrity.The results of Nissl staining showed that AQP4 knockout did not affect the size and morphology of lateral ventricles,and the morphology and number of neurons did not change significantly.（3）The results of Sagittal immunofluorescence showed that the glymphatic system disorder caused the increase ofαSyn expression in the whole brain.（4）The glymphatic system dysfunction leads to early PD-like manifestations such as decreased olfactory function,prolonged pole climbing time,and increased autonomous activities in two-month-old mice;（5）As the consistent glymphatic pathway dysfunction,pathological phosphorylationαSyn was significantly increased in the brain of two-month-old and twenty-month-old mice,and the number of dopaminergic neurons was significantly decreased in twenty-month-old mice.（6）h SNCA^fl/fl;Nestin^Cretransgenic mice overexpressing human-derivedαSyn in the whole brain,and there were no significant behavioral changes in three-month-old and twelve-month-old mice;（7）Compared with wild-type mice,the polarity of AQP4 in the midbrain and striatum of A53T model mice was reduced,and the fluorescence intensity was increased,but there was no significant change in the expression of AQP4 in the cortex and hippocampus;（8）Compared with h SNCA^fl/flmice,h SNCA^fl/fl;Nestin^Cretransgenic mice had lower polarity of AQP4 in the midbrain and striatum,and protein expression of AQP4 was increased,but the cortex and hippocampus AQP4 expression has no significant changes;（9）Compared with h SNCA^fl/flmice,h SNCA^fl/fl;Nestin^Cretransgenic mice with AZA continuous administration have increased expression ofαSyn and phosphorylatedαSyn in the midbrain and striatum;（10）Compared with h SNCA^fl/flmice,h SNCA^fl/fl;Nestin^Cretransgenic mice have decreased expression of SYP38 in the striatum after AZA continuous administration.Conclusion:The glymphatic system is involved in regulating the clearance of centralαSyn.Overexpression ofαSyn affects the function of AQP4.The glymphatic pathway dysfunction andαSyn accumulation affect each other and accelerate the PD-like pathological process in mice.In summary,the main innovations of our work are listed as follows:1.Disorders of the glymphatic system induced by long-term AQP4 deletion can induce accumulation of pathologicalαSyn and loss of dopaminergic neurons.2.Overexpression ofαSyn will damage the AQP4 polarity in the midbrain and striatum.After AZA modeling,it will aggravate the accumulation of pathologicalαSyn caused by glymphatic system dysfunction.3.The glymphatic system dysfunction can increase the expression of the pathologicalαSyn,induce behavioral disorders,and injure DA neurons in the mouse brain,indicating that the glymphatic system regulates the pathological process of PD.It suggests that glymphatic system dysfunction andαSyn accumulation are a vicious cycle process,which provides ideas for developing PD treatment drugs targetingαSyn.