The role of autophagy in TLR2-mediated alpha-synuclein accumulation in neurons.

Abstract

Parkinson’s disease (PD) is one of the most common neurodegenerative disorders in the world. It is pathologically characterized by a loss of dopaminergic neurons and accumulation of protein inclusions called Lewy Bodies (LBs). α-Synuclein (α-syn) is one of the most prominent proteins is LBs and can aggregate into large oligomers and fibrils that might be neurotoxic. Defects in protein clearance pathways such as the autophagy lysosomal pathway (ALP) might be a contributing factor to α-syn pathology. Toll-like receptor 2 (TLR2) activation inhibits autophagy in neurons in vitro. In this project, I aimed to further determine the role of TLR2 signaling in regulating the ALP and subsequent α-syn accumulation in neurons. TLR2 activation caused an increase in autophagy markers followed by an accumulation of α-syn. TLR2 activation also potentiated the α-syn accumulation caused by seeding neurons with α-syn pre-formed fibrils (PFFs). This potentiation could be rescued genetically by TLR2-knockout and pharmacologically by inhibition of TLR2 with a small molecule antagonist. Following the results obtained in the neurons, the next aim was to determine the effect of TLR2 signaling in peripheral immune cells. In macrophages, autophagy inhibition was measured upon TLR2 activation. However, α-syn was not seen to accumulate macrophages. Interestingly, upon TLR4 activation with lipopolysaccharide (LPS), the expression levels of α-syn in macrophages was significantly decreased. This was accompanied by an increase in the release of α-syn from immune cells into the extracellular space. In line with the interest in measuring the ALP in neurons, my next aim was to develop a high-content screening toolkit using imaging cytometry, to measure and quantify ALP phenotype in cortical neurons. Finally, the developed HCS workflow was used to determine the importance of PD related mutations on the ALP.