Targeting and Localization of Alpha-Synuclein in Primary Neurons

Axonal transport dysfunction and aberrant accumulation of alpha-synuclein (alpha-syn) has been linked to neurodegenerative conditions such as Parkinson's disease (PD) and dementia with Lewy bodies. Previous studies have shown that alpha-syn normally undergoes delayed localization to presynaptic terminals but the key structural elements involved have not been fully characterized nor the trafficking behavior well understood. Conflicting reports exist as to whether the protein moves predominantly by fast or slow component routes. Here we describe the axonal movement and presynaptic distribution of various GFP-tagged alpha-syn constructs in vitro using live fluorescence microscopy. Earlier in differentiation (10-14DIV), the protein was clearly tracked to travel bidirectionally at a heterogeneous rate in concentrated punctate structures. Later in differentiation (17-21DIV), when normal concentration at the presynapse occurs, the N-terminal deletion exons 2, 3, and 4 mutants failed to localize completely, suggesting those exons which comprise the helical domain of the protein to be necessary for normal targeting to the presynapse. In addition, familial PD-associated mutants A30P and A53T demonstrate reduced localization and transport velocity, respectively, compared to wildtype controls. This is the first report of the effects of exon-based deletions on the dynamic behavior of alpha-syn in live cultures of primary neurons. Further examination of the subcellular localization of alpha-syn through a novel correlated light and electron microscopy technique, revealed alpha-syn to bind to vesicular and membranous structure at the presynaptic terminal. Finally, analysis of alpha-syn retention in digitonin-permeabilized cells indicate that a subpopulation of alpha-syn associates with plasma membrane, but contrary to published reports, this plasma membrane component does not have characteristics consistent with lipid rafts.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Cell and Developmental Biology

Grantor

University of Illinois at Urbana-Champaign

Year dc:date

2015

Author dc:creator

• Yang, Mong-Lin

Contributors dc:contributor

• Kemper, Byron W.
• George, Julia M.

• Biology, Cell

Language dc:language

eng

Identifier

(MiAaPQ)AAI3363118

OAI identifier oai:identifier

oai:www.ideals.illinois.edu:2142/86326