Investigation On Molecular Mechanisms Of Synphilin-1 Aggregate Formation In Saccharomyces Cerevisiae

Parkinson’s disease(PD)is one of the most prevalent neurodegenerative diseases and the presence of cytoplasmic inclusions is known as a typical hall marker of this disease.Synphilin-1 as one of the major components of PD inclusion bodies interacting with several proteins involved in PD pathogenesis imply that it plays a key role in inclusion body formation.However,the molecular mechanisms of synphilin-1 aggregate formation and physiological function of synphilin-1 remain unclear due to limition of studys on aggregation process and complexity of genome in human and mammalian models.Yeast(Saccharomyces cerevisiae)has been proved as a powerful model on research of human disease due to its own advantages.Most of yeast genes have robust human homologues or conserved domain with human genes,and genomic homology explains the conservation of fundamental cell biological processes between yeast and mammalian cells,including protein aggregation.Therefore,we aimed to decipher the genes and pathway involved in presumed human disease protein synphilin-1 aggregate formation in Saccharomyces cerevisiae by a genome-wide,high-content imaging based screening approach(HCI)and the yeast synthetic genetic array(SGA)technology.The enrichment and interaction network analysis for these genes were applied by bioinformatics to further investigate the molecular mechanisms of synphilin-1 aggregate formation.To screen the genes involved in synphilin-1 aggregate formation,yeast query strains Y7092-285 were constructed for synphilin-1 by transforming the Y7092 background strain with a high-copy plasmid containing the synphilin-1 gene fused to DsRed gene.Based on the query stain,both yeast single gene knock-out collection and essential gene temperature-sensitive allele collection with synphilin-1-DsRed expressing were also established using the yeast SGA technology,named S2YDSY1.To identify the mutants displayed a synthetic sick when expressing synphilin-1,a growth defect screening was performed.Combined the software analysis and verification manually,57 mutants were identified.Functional enrichment and network interaction analysis suggested that 4 functional categories including microtubule-based transport,membrane lipid biosynthetic process,THO complex and Prefoldin complex are required for maintaining the normal growth of cells under synphilin-1 expression.To identify genes and cellular components that regulate synphilin-1 aggregation with unexpected functionalities,we developed and validated a yeast HCI screening approach for the fluorescently tagged human synphilin-1 aggregates.By combining with a secondary screening for mutants showing significant changes on fluorescence signal intensity,we filtered out hits that significantly decreased the expression level of synphilin-1.We found 133 yeast genes that didn’t affect synphilin-1 expression but that were required for the formation of synphilin-1 aggregates.Functional enrichment and physical interaction network analysis revealed these genes to encode for functions involved in cytoskeleton organization,histone modification,sister chromatid segregation,glycolipid biosynthetic process,and DNA replication.To further clarify the molecular mechanism of synphilin-1 aggregate formation,DYN3,GIM4,KAR3,NUM1,PAC10,SHE4,YKE2,VAC14,ACT1,GPI19 genes were identified to be required for synphilin-1 aggregate formation and detoxification of cells with synphilin-1 expression,based on the analysis of screening for both the growth defect and aggregate formation.Further growth assays of these mutants showed a significant synthetic sick effect upon synphilin-1 expression,which supports the hypothesis that mature aggregates/inclusions represent an end stage of several events that have a protection effect against the synphilin-1 cytotoxicity.These especially genes with homologues in human,provided valuable clues concerning the machinery controlling synphilin-1 aggregate formation and pathogenesis of PD.Utilizing cell stains and immunofluorescence technique,it identified the co-localization of synphilin-1 aggregates with mitochondria and actin cytoskeleton.Moreover,the synphilin-1 aggregates were recognized to accumulate in both juxtanuclear quality control compartment and insoluble protein deposit compartment.It was supposed that synphilin-1 aggregates were mitochondria-tethered,and the aggregate formation was based on fixation and transport of actin cytoskeleton,regulated by the modification of histones and glycolipid biosynthetic pathway in yeast cells.These results acquired from the yeast model gave an unbiased global view on the complexity of the machinery underlying aggregate formation by synphilin-1 and provided valuable clues and theoretical basis concerning the machinery controlling synphilin-1 aggregate formation.Further investigation of these human homologue hits can provide novel insights for our understanding of the mechanisms of pathogenesis causing Parkinson’s disease as well as other protein folding disorders.