THE FORMATION, EXPANSION AND DISSOLUTION OF A-SYNUCLEIN INCLUSIONS скачать в хорошем качестве

THE FORMATION, EXPANSION AND DISSOLUTION OF A-SYNUCLEIN INCLUSIONS

Talk by Eleanna Kara Rutgers–New Brunswick, New Brunswick, USA of America at the 23rd HFSP Awardees Meeting (17-19 June 2024, Washington DC, USA) Abstract Introduction: α-synuclein (αSyn) accumulates in the brains of patients with Parkinson’s disease (PD) and forms inclusions in neurons called Lewy Bodies (LBs). LBs initially form in the brainstem and, as the disease progresses, are also observed in rostral regions. It has been hypothesized that this apparent spread of pathology is caused by the prion-like cell-to-cell transfer (propagation) of αSyn. Results: To understand the mechanism underlying the propagation of αSyn, we did a high throughput screen. We cloned a construct encoding GFP-T2A-αSyn-RFP, with which we transiently transfected a HEK QBI cell line overexpressing wild type αSyn. The translated protein is cleaved at the 2A position, thus producing two independent proteins: GFP and αSyn-RFP. The latter then transfers to neighboring cells and the populations can be identified based on their colors: donor cells are RFP+GFP+ and recipient cells are RFP+GFP-. We used this model system to complete a genome wide, imaging based, arrayed siRNA high throughput screen that identified 38 genes whose knock down modifies the propagation of αSyn. Several of those genes have been implicated in the pathogenesis of neurodegeneration. Weighted gene coexpression network analysis (WGCNA) using gene expression data from multiple regions from healthy human brains showed that the 38 genes co-cluster with known PD genes in the same gene expression modules more frequently than expected by random chance. Follow up experiments in a novel tissue culture model system showed that αSyn molecules are mobile within inclusions as tested by FRAP experiments. This is consistent with the inclusions being liquid condensates formed via phase separation. Knock-down of two of the 38 genes increases the number and decreases the size of αSyn inclusions by modulating phase separation. One of those genes is also involved in pathways regulating the biogenesis of lipid droplets, and its knock-down affects the co-condensation between αSyn and lipid droplets. Experiments in iNeurons and molecular dynamic simulations are in progress to determine whether the underlying protein networks are enriched in intrinsically disordered proteins, and whether dysregulation of phase separation on a larger scale is the common mechanism underlying αSyn dysregulation in PD. Conclusion: Our findings suggest that there is a link between αSyn aggregation, propagation and phase separation, and underlying pathways are regulated by genetic networks that dysfunction in PD.