An investigation into the toxic mechanism of alpha-synuclein: implications for Parkinson’s disease therapeutics

© 2018 Dr. Stephanie Jane GuineyParkinson’s disease is a neurodegenerative disorder that causes motor, cognitive and neuropsychiatric deficits in patients. Current therapies do not stop ongoing neurodegeneration, and investigating biochemical features present in brain regions affected by cell death may enable identification of mediators of neurodegeneration to identify targets for potential disease-modifying drugs. α-synuclein is a protein found aggregated in Lewy bodies, a major pathology present in patients’ brains, and mutations and variants in the α-synuclein-encoding gene can increase susceptibility to Parkinson’s disease. Observations from human post-mortem studies fuelled a hypothesis that α-synuclein pathology may spread between adjacent brain regions and contribute to neurodegeneration, which were further supported by animal studies utilising injection of aggregated α-synuclein into the brain. The purpose of this thesis was to optimise a cell culture model to test the mechanism of α-synuclein toxicity. In multiple cell types, I applied aggregated α-synuclein pre-formed fibrils (PFFs), to model toxic pathways activated by pathological α-synuclein. Since iron levels are elevated in patients’ brains, and improvement in patient’s motor function was observed in response to iron chelation therapy in a phase II clinical trial, I hypothesised that α-synuclein PFFs might cause toxicity via ferroptosis, an iron- and lipid peroxidation-dependent cell death pathway identified in 2012 that warrants investigation as a cause of cell death in Parkinson’s disease. I addressed this hypothesis using a predominantly pharmacological approach in which drugs known to inhibit or exacerbate ferroptosis were added to cells prior to α-synuclein PFFs, and the effect of each drug on toxicity was compared to that of ferroptosis inducer, erastin. Toxicity of α-synuclein was inhibited by liproxstatin-1, a classic anti-ferroptotic agent, but was otherwise not impacted by a range of other ferroptosis inhibitors or inducers. Lipid peroxide levels, which elevate during ferroptosis, were not changed following administration of α-synuclein PFFs, indicating that liproxstatin-1 likely reduced α-synuclein toxicity via a ferroptosis-independent mechanism. Liproxstatin-1 concentrations that were protective against α-synuclein toxicity caused similar elevation of autophagy-lysosomal pathway markers as known lysosomal acidification inhibitors, chloroquine and bafilomycin A1. This highlighted lysosomal inhibition as a potential mechanism by which α-synuclein PFF toxicity might be reduced. Both chloroquine and bafilomycin, and inhibitors of lysosomal cysteine proteases, leupeptin and E-64D, significantly recovered loss of viability in α-synuclein-treated cells. In parallel confocal imaging analysis, I observed that while the majority of α-synuclein remained outside of the cell, a small proportion entered the endo-lysosomal pathway; furthermore, chloroquine treatment did not prevent PFF association with this pathway. In contrast, heparin also protected against α-synuclein toxicity, and prevented membrane association and cellular internalisation of α-synuclein PFFs. I concluded that the toxicity of α-synuclein was likely caused by internalised α-synuclein associated with the lysosome, with heparin reducing toxicity by interfering with α-synuclein uptake. The protection conferred by lysosomal inhibitors may not involve preventing α-synuclein uptake into the lysosome but suggested that aggregated α-synuclein requires a functional lysosome to initiate toxicity. Thus preventing aggregated α-synuclein from entering cells, or inhibiting a lysosomal function critical for its toxicity should be an aim for future Parkinson’s disease therapies