In Vivo Assessment of Alpha-Synuclein Secretion

Objective/Rationale: 
Recent data from our lab (Emmanouilidou et al, 2010) suggest that cell-produced physiologically secreted alpha-synuclein can cause the death of neuronal recipient cells. Although the pathophysiological role of the secreted alpha-synuclein forms remain largely unknown we have found that alpha-synuclein secretion depends on intracellular calcium concentrations and that involves exosomes. Using a highly sensitive, in-house ELISA, and in vivo microdialysis, we intend to examine the effects of pharmacological manipulation of specific cellular pathways on the secretion of alpha-synuclein. We are also planning to investigate the neuronal sites from which alpha-synuclein is secreted.

Project Description: 
We will pharmacologically manipulate two fundamental non-classical secretory pathways, namely the ABC transporter-based and the exosome-based pathways using specific compounds administered locally in pre-clinical models through dialysis membrane.

Following guided cannula and probe implantation in the pre-clinical model, ISF will be collected hourly for 8 hours. For alpha-synuclein detection and quantification, microdialysis samples will be analysed by a novel ultra-sensitive ELISA assay.

In the second part of the project, we will use custom made cell-culture chambers in an attempt to segregate neurons into somal and neuritic compartments. Alpha-synuclein in the different compartments will be measured with the newly developed ELISA.

Relevance to Diagnosis/Treatment of Parkinson’s Disease:  
Secreted alpha-synuclein may be a hitherto unrecognized agent that contributes to PD pathogenesis. It is possible that alpha-synuclein secretion serves to amplify and propagate PD-related pathology in a paracrine manner. We have found using in an in vitro cell model that alpha-synuclein is normally secreted via a non-classical exosomal pathway. Our new in vivo approach gives us the opportunity to rationally test agents that have been shown to manipulate specific cellular pathways involved in non-classical protein secretion. Our goal is to gain further insight into the mechanisms that affect alpha-synuclein levels extracellularlly and thus identify new therapeutic targets for PD.

Anticipated Outcome: 
The current proposal will help investigate mechanisms of synuclein secretion in vivo. We anticipate that our work will shed light on the physiological mechanisms of alpha-synuclein secretion and understand how disregulation of such pathways may be linked to the pathogenesis of PD.