Mapping the Subcellular Distribution of α-Synuclein in Neurons using Genetically Encoded Probes for Correlated Light and Electron Microscopy: Implications for Parkinson's Disease Pathogenesis

Modifications to the gene encoding human alpha-synuclein have been linked to the development of Parkinson's disease. The highly conserved structure of alpha-synuclein suggests a functional interaction with membranes, and several lines of evidence point to a role in vesicle-related processes within nerve terminals. Using recombinant fusions of human alpha-synuclein, including new genetic tags developed for correlated light microscopy and electron microscopy (the tetracysteine-biarsenical labeling system or the new fluorescent protein for electron microscopy, MiniSOG), we determined the distribution of alpha-synuclein when overexpressed in primary neurons at supramolecular and cellular scales in three dimensions (3D). We observed specific association of alpha-synuclein with a large and otherwise poorly characterized membranous organelle system of the presynaptic terminal, as well as with smaller vesicular structures within these boutons. Furthermore, alpha-synuclein was localized to multiple elements of the protein degradation pathway, including multivesicular bodies in the axons and lysosomes within neuronal cell bodies. Examination of synapses in brains of transgenic mice overexpressing human alpha-synuclein revealed alterations of the presynaptic endomembrane systems similar to our findings in cell culture. Three-dimensional electron tomographic analysis of enlarged presynaptic terminals in several brain areas revealed that these terminals were filled with membrane-bounded organelles, including tubulovesicular structures similar to what we observed in vitro. We propose that alpha-synuclein overexpression is associated with hypertrophy of membrane systems of the presynaptic terminal previously shown to have a role in vesicle recycling. Our data support the conclusion that alpha-synuclein is involved in processes associated with the sorting, channeling, packaging, and transport of synaptic material destined for degradation.