Establishment Of Screening Platforms For Compounds That Promote The Dissociation Or Degradation Of α-synuclein Oligomers And The Researches On Compounds FY-017/CA In Anti-parkinson’s Disease

Parkinson’s disease(PD)is the second largest neurodegenerative disease after Alzheimer’s disease.A53T mutant ofα-synuclein was found to be the first disease-related protein with Parkinson’s disease in 1997.A53T mutant ofα-synuclein increases the neurocytoxicity with misfolding and forming more toxic aggregates.Numerous studies have shown that the deposition of Lewy bodies withα-synuclein as the main component is also detected in the brain with sporadic PD.Theα-synuclein abnormality plays an important role in the early onset and the development of the disease,and it is found that the abnormal aggregation ofα-synuclein appeared earlier than the typical symptoms of PD,inhibitingα-synuclein aggregation should be able to slow the progression of PD and improve symptoms.α-synuclein mutants or oligomers can be used as a clinical biomarker at the early stage of PD pathogenesis.Finding more compounds that can effectively reduce A53Tα-synuclein oligomers is of vital importance to treat Parkinson’s Disease.We used a luciferase-based protein-fragment complementation assay(PCA)and bi-molecular fluorescence complementation(BiFC)method to construct a cell model expressing a fusion protein containing A53Tα-synuclein and Gaussia luciferase fragments,then to screen hit compounds that promote the dissociation or degradation of A53Tα-synuclein oligomers,at a high-throughput efficiency with the Z’factor 0.62.In addition,the cell model that expresses fusion proteins containing A53Tα-synuclein and Venus fragments was used to observe the effects of compounds on A53Tα-synuclein oligomers directly.Furthermore,FY-017 significantly enhanced cell viability and attenuated MPP~+-induced cell damage demonstrated in SH-SY5Y.We have used a classic MPTP acute Parkinson mouse model and A53Tα-synuclein transgenic PD mouse model to evaluate the pharmacodynamics of FY-017 in vivo.The results showed that FY-017 significantly improved the motor dysfunction of PD model mice and the degeneration of dopaminergic neurons in the nigrostriatal detected with behavioural methods and immunohistochemical staining,respectively.These studies provide a scientific basis for FY-017 as a candidate drug to treat Parkinson’s disease.To further explore the mechanism by which the candidate compound FY-017promotes the depolymerization or degradation of A53Tα-synuclein oligomers,we found that FY-017 could reduce the aggregation of A53Tα-synuclein effectively by THT protein aggregation assay.Western blotting assay showed that A53Tα-synuclein was reduced with FY-017 treatment.A53Tα-synuclein as misfolded protein is mainly degraded by the autophagic lysosomal system.mTOR is a switch protein of the autophagy pathway,and inhibition of mTOR promotes autophagy in cells.We verified that FY-017 could activate mTOR-mediated autophagy to promote the degradation of A53Tα-synuclein.The autophagy inhibitor bafilomycin A1 and mTOR agonist MHY-14885 could reverse the efficacy of FY-017.Therefore,targetting the regulation of autophagy degradation ofα-synuclein or mTOR inhibitor provides a new therapeutic strategy for the drug development of anti-Parkinson’s disease.Caffeic Acid(CA)has shown neuroprotective effects in Alzheimer’s disease or cerebral ischemia models;CA Tablets could be as a Second-line Therapy for Immune Thrombocytopenia in clinic.New use of old drugs can speed up the development of new drugs.However,it is unclear whether CA has neuroprotective effects in Parkinson’s disease.First,we found that CA could reduce A53Tα-synuclein oligomers by PCA screening platform and alleviate cell damage induced by overexpression of A53Tα-synuclein.It was furtherly demonstrated that CA did not affect the transcription or secretion ofα-synuclein in a cell model overexpressing A53Tα-synuclein by reverse transcription real-time quantitative PCR and Elisa,but promoted protein degradation of A53Tα-synuclein detected with western blotting.JNK is an upstream regulatory element of autophagy,and JNK regulates its binding to Beclin-1 by phosphorylating Bcl-2 protein to regulate the formation of autophagic membrane.We found that CA degraded A53T alpha-synuclein by activating JNK/Bcl-2 mediated autophagy.The autophagy inhibitor bafilomycin A1 and the JNK inhibitor SP600125 reversed the effects of CA.Finally,in A53Tα-synuclein transgenic mice,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and probenecid(MPTP/p)mice,rotarod test,pole test,open field,catwalk gait analysis,buried food pellet test,and immunohistochemical staining,western blotting were used to evaluate the neuroprotective effect of CA.The results showed that CA not only improved motor disorders and olfactory dysfunction but also reduced the damage of dopaminergic neurons and decreasedα-synuclein in the substantia nigra.This study firstly proposes a neuroprotective effect of CA-targeted JNK/Bcl-2-mediated autophagy to degrade pathologicalα-synuclein protein in Parkinson’s disease.This work has established a high-throughput screening platform to screen out hit compounds that promoting the depolymerization or degradation of A53Tα-synuclein oligomers.These work provides research evidence for drug development targeting autophagy to degradeα-synuclein in Parkinson’s disease,and lays the foundation for small molecule compound FY-017’s and CA’s clinical trials in future.