| Abstract [eng] |
Parkinson's disease is a neurodegenerative disorder characterized by insoluble α-syn deposits called Lewy bodies. The protein aggregation process into soluble aggregates and fibrils is considered to be the disease causative mechanism. Recent studies show that protein aggregates are toxic to neurons and can induce activation of glial cells (Ingelsson, 2016b). However, it is still unknown which α-syn forms are the most neurotoxic. This study aimed to investigate the effects of the different α-syn aggregates on brain cells. The objectives were: 1) to prepare protocols on α-syn self-aggregation and aggregation using Aβ1-42 oligomers; 2) to characterize α-syn aggregates using the atomic force microscopy; 3) to evaluate α-syn induced neurotoxicity in primary neuronal-glial cell cultures; 4) to evaluate whether Aβ1-42 has an impacat on α-syn oligomerization and toxicity. For the preparation of α-syn aggregates, seven protocols were used. Monomeric protein was incubated for 24, 48 hours, and 7 days at room temperature with and without aggregation inductor Aβ1-42 (140:1 α-syn: Aβ1-42). The degree of α-syn oligomerization was established using atomic force microscopy (AFM). Experiments on α-syn cytotoxicity were carried out using primary neuronal-glial cell cultures. Cell viability and number were evaluated by fluorescence microscopy. Cellular nuclei were stained with propidium iodide and Hoechst33342, and microglial cells were labelled using Isolectin-IB4 conjugated with AlexaFluor488. The results show that after 24 and 48 hours (± Aβ) of incubation, α-syn aggregates observed by AFM were spheric oligomers, and after 7-day (± Aβ) incubation, a protein formed spheric oligomers and fibrils. Co-incubation with Aβ1-42 promotes the formation of larger oligomers but smaller fibrils, characterized by z-axis. We found that a medium 7-35 nm oligomers, formed after 24 h incubation with Aβ1-42, exert more pronounced neurotoxic effects at lower 3 μM and higher 9 μM concentrations. These oligomers induced neuronal death via necrosis. Microglial and astrocyte proliferation was caused by oligomeric and fibrillar species formed after 48 hours or 7 days of incubation with or without Aβ1-42. However, at higher concentration (9 μM) alpha-synuclein aggregates caused astrocyte proliferation while did not affect microglial number. Taken together, our results show that the effect of α-syn species on cell viability and the number of neurons and glial cells depends on particle size, concentration, and incubation with CGC time. |
