Objective/Rationale:
Extracellular alpha-synuclein appears to be central in the pathogenesis, and perhaps progression of PD. The mechanism(s) underling alpha-synuclein secretion are important to be elucidated since they may represent a common, but as yet unidentified, disease mechanism. We have established a reverse microdialysis approach in conjuction with a novel, ultra-sensitive synuclein ELISA that would allow us to pharmacologically manipulate distinct secretory pathways in a pre-clinical striatum. Such a methodology will distinguish possible pathways involved in the secretion of alpha-synuclein in vivo.
Project Description:
To identify the mechanisms involved in alpha-synuclein secretion in vivo, we will pharmacologically target known secretory pathways in the striatum by the local administration of specific compounds using reverse microdialysis. Detection of alpha-synuclein in the interstitial fluid of the brain will be accomplished by an ultra-sensitive, in house ELISA. More specifically, we will investigate whether classical Ca2+-regulated exocytosis, ABC transporters / channels, and exosome secretion are implicated in alpha-synuclein release in vivo. Furthermore, we plan to address a role of exosomes in the observed neurotoxicity induced by extracellular alpha-synuclein. To this end, we will investigate the effects of exosome-associated alpha-synuclein on the integrity of the axonal network. This will be achieved by using primary compartmentalized neuronal cultures using microfluidic culture devices.
Relevance to Diagnosis/Treatment of Parkinson’s Disease:
Our data so far adds to the suggestion that mechanisms involved in the regulation of alpha-synuclein release may underlie a common pathway in the disease pathogenesis and as such may be targets to modify PD progression. This proposal will investigate the specific pathways that underpin the release of alpha-synuclein in vivo and help us understand the factors that may be involved in the spread of alpha-synuclein pathology in PD. We believe that this information will enable us to design and test possible therapies to influence the release and perhaps transfer of alpha-synuclein between cells which may be useful strategies for PD treatment.
Anticipated Outcome:
We have successfully established a reverse microdialysis system to identify potential regulators of alpha-synuclein secretion in vivo in the brain. We anticipate that the proposal results will aid in the characterization of novel mechanisms involved in the regulation of extracellular alpha-synuclein levels and its function in the progression of PD.
Final Outcome
To elucidate the mechanism of alpha-synuclein secretion in a model, we used reverse microdialysis to locally administrate in a pre-clinical model striatum compounds that trigger specific intracellular pathways. We have found that, in the striatum of the pre-clinical model alpha-synuclein release is a calcium-dependent process that seems to be regulated by the crosstalk between GABAergic and glutamatergic neurons. We are also investigating whether, and to what extent, alpha-synuclein is released in association with exosomes and whether exosome-associated alpha-synuclein affects recipient neurons. Our data suggest that alpha-synuclein is associated with exosomes isolated from the culture medium of primary neurons in a manner that follows the levels of alpha-synuclein expression. Using these exosomes, we have found that recipient neurons can readily uptake exosomes by endocytosis. We are in the process of investigating whether this uptake affects any aspect of neuronal homeostasis.