Objective/Rationale:
The pathophysiological roles of the secreted AS forms remain largely unknown. We have identified oligomeric and monomeric AS species in the conditioned medium (CM) of SHSY5Y cells inducibly expressing WT AS. This CM is toxic to recipient neuronal cells. Interestingly, cell death is correlated with the concentration of AS and the presence of oligomeric AS species in the CM. We are planning to investigate whether AS is actually secreted at biologically meaningful concentrations and the effects it exerts extracellularly in mouse brain.
Project Description:
In the first part of the project, we intend to examine whether AS is naturally secreted in mouse brain. For this purpose, we are going to use in vivo microdialysis in double transgenic mice expressing human A53T AS. Following guided cannula and probe implantation in the mouse cortex, CSF will be collected hourly for 8 hours. For AS detection and quantification, microdialysis samples will be analysed by a sensitive ELISA assay.
In the second part of the project, we will use size exclusion chromatography to isolate naturally secreted oligomeric and monomeric AS species from the CM of SHSY5Y cells inducibly expressing WT AS. We hypothesize that these species possess different biological activities, and we intend to determine their effects on synapses and dendritic spines in mouse acute brain slices containing the substantia nigra and striatum.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Secreted AS may be a hitherto unrecognized agent that contributes to PD pathogenesis. It is possible that AS secretion serves to amplify and propagate PD-related pathology in a paracrine manner. We plan to put to the test the theory of AS toxicity via soluble species in the setting of physiologically secreted AS in vivo. Our approach gives us the opportunity to rationally test agents that have been shown to diminish AS toxicity.In addition, quantification of AS in biological fluids may in the future serve as a biomarker for PD.
Anticipated Outcome:
The current proposal will help establish a novel microdialysis approach to effectively quantify AS present in the interstitial fluid of mouse brain. We anticipate that our work will shed light on the existence of naturally secreted AS and its effects on neuronal cell homeostasis.
Final Outcome
Alpha-synuclein has long been considered an intracellular protein. However, recent reports have suggested that the molecule is present in biological fluids of humans and in the culture medium of neuronal cells. Still, the question remains whether alpha-synuclein can be secreted physiologically. We sought to provide evidence of alpha-synuclein secretion in vivo. We have developed highly sensitive methodology and found that alpha-synuclein is present in the brain parenchyma of mice and humans. Additionally, we have investigated the effects of secreted synuclein on neurite homeostasis. Our data suggest that alpha-synuclein is physiologically secreted and may have paracrine actions. It is possible that fluctuations in the levels of extracellular alpha-synuclein may contribute to the spread of pathology in PD. As such, extracellular alpha-synuclein can be considered a target for therapeutic intervention in PD.