Progressive accumulation of α-synuclein is a key step in the pathological development of Parkinson’s disease. Impaired protein degradation and increased levels of α-synuclein may trigger a pathological aggregation in vitro and in vivo. The chaperone-mediated autophagy (CMA) pathway is involved in the intracellular degradation processes of α-synuclein. Dysfunction of the CMA pathway impairs α-synuclein degradation and causes cytotoxicity. In the present study, we investigated the effects on the CMA pathway and α-synuclein aggregation using bioactive ingredients (Dihydromyricetin (DHM) and Salvianolic acid B (Sal B)) extracted from natural medicinal plants. In both cell-free and cellular models of α-synuclein aggregation, after administration of DHM and Sal B, we observed significant inhibition of α-synuclein accumulation and aggregation. Cells were co-transfected with a C-terminal modified α-synuclein (SynT) and synphilin-1, and then treated with DHM (10 μM) and Sal B (50 μM) 16 hours after transfection; levels of α-synuclein aggregation decreased significantly (68% for DHM and 75% for Sal B). Concomitantly, we detected increased levels of LAMP-1 (a marker of lysosomal homeostasis) and LAMP-2A (a key marker of CMA). Immunofluorescence analyses showed increased colocalization between LAMP-1 and LAMP-2A with α-synuclein inclusions after treatment with DHM and Sal B. We also found increased levels of LAMP-1 and LAMP-2A both in vitro and in vivo, along with decreased levels of α-synuclein. Moreover, DHM and Sal B treatments exhibited anti-inflammatory activities, preventing astroglia- and microglia-mediated neuroinflammation in BAC-α-syn-GFP transgenic mice. Our data indicate that DHM and Sal B are effective in modulating α-synuclein accumulation and aggregate formation and augmenting activation of CMA, holding potential for the treatment of Parkinson’s disease.

Although converging evidence point to alpha-synuclein (a-syn) aggregation and Lewy body (LB) formation as central events in Parkinson's disease (PD), the molecular mechanisms that regulate these processes and their role in disease pathogenesis remain poorly understood. Herein, we applied an integrative biochemical, structural and imaging approach to elucidate the sequence, molecular and cellular mechanisms that regulate LB formation in primary neurons. Our results establish that post-fibrillization C-terminal truncation mediated by calpains 1 and 2 and potentially other enzymes, plays critical roles in regulating a-syn seeding, fibrillization and orchestrates many of the events associated with LB formation and maturation. These findings combined with the abundance of a-syn truncated species in LBs and pathological a-syn aggregates have significant implications for ongoing efforts to develop therapeutic strategies based on targeting the C-terminus of a-syn or proteolytic processing of this region.

The formation of α-synuclein aggregates is a major pathological hallmark of Parkinson's disease. Copper promotes α-synuclein aggregation and toxicity in vitro. The level of copper and copper transporter 1, which is the only known high-affinity copper importer in the brain, decreases in the substantia nigra of Parkinson's disease patients. However, the relationship between copper, copper transporter 1 and α-synuclein pathology remains elusive. Here, we aim to decipher the molecular mechanisms of copper and copper transporter 1 underlying Parkinson's disease pathology. We employed yeast and mammalian cell models expressing human α-synuclein, where exogenous copper accelerated intracellular α-synuclein inclusions and silencing copper transporter 1 reduced α-synuclein aggregates in vitro, suggesting that copper transporter 1 might inhibit α-synuclein pathology. To study our hypothesis in vivo, we generated a new transgenic mouse model with copper transporter 1 conditional knocked-out specifically in dopaminergic neuron. Meanwhile, we unilaterally injected adeno-associated viral human-α-synuclein into the substantia nigra of these mice. Importantly, we found that copper transporter 1 deficiency significantly reduced S129-phosphorylation of α-synuclein, prevented dopaminergic neuronal loss, and alleviated motor dysfunction caused by α-synuclein overexpression in vivo. Overall, our data indicated that inhibition of copper transporter 1 alleviated α-synuclein mediated pathologies and provided a novel therapeutic strategy for Parkinson's disease and other synucleinopathies.

Aggregated α-synuclein (α-syn) is the main constituent of Lewy bodies, which are a pathological hallmark of Parkinson’s disease (PD). Environmental factors are thought to be potential triggers capable of initiating the aggregation of the otherwise monomeric α-syn. Braak’s seminal work redirected attention to the intestine and recent reports of dysbiosis have highlighted the potential causative role of the microbiome in the initiation of pathology of PD. Staphylococcus aureus is a bacterium carried by 30–70% of the general population. It has been shown to produce functional amyloids, called phenol soluble modulins (PSMαs). Here, we studied the kinetics of α-syn aggregation under quiescent conditions in the presence or absence of four different PSMα peptides and observed a remarkable shortening of the lag phase in their presence. Whereas pure α-syn monomer did not aggregate up to 450 h after initiation of the experiment in neither neutral nor mildly acidic buffer, the addition of different PSMα peptides resulted in an almost immediate increase in the Thioflavin T (ThT) fluorescence. Despite similar peptide sequences, the different PSMα peptides displayed distinct effects on the kinetics of α-syn aggregation. Kinetic analyses of the data suggest that all four peptides catalyze α-syn aggregation through heterogeneous primary nucleation. The immunogold electron microscopic analyses showed that the aggregates were fibrillar and composed of α-syn. In addition of the co-aggregated materials to a cell model expressing the A53T α-syn variant fused to GFP was found to catalyze α-syn aggregation and phosphorylation in the cells. Our results provide evidence of a potential trigger of synucleinopathies and could have implications for the prevention of the diseases.