Analysis of Aggregate Formation of Mutant Forms of Alpha Synuclein
Human familial Parkinson's disease (PD) is characterized by an A53T mutation of the neural protein alpha synuclein (AS) which aggregates under oxidative stress conditions. Despite normally having a theonine residue at position 53 of AS, wild-type mice do not appear to develop PD. Other variations in murine AS are suspected to contribute to aggregation resistance, particularly the variation at S87N located in the protein's highly amyloidogenic portion. This variation (S87N) in murine AS is predicted to prevent wild-type AS in mice from demonstrating neurotoxicity even when fibrillized. Tendency of AS to aggregate was tested under experimental oxidative stress conditions on two transfected lines of a cell model of PD (BE-M17), mutant A53T and double-mutant A53TS87N. Florescent microscopy images suggested that aggregation in A53TS87N transfected cells under oxidative stress were approaching a five-fold lower aggregate percentage than A53T mutants. Additionally, immunoblotting of lysates from each cell line showed a much lower concentration of aggregate-prone high molecular weight AS and a higher concentration of low molecular weight AS in the double mutant (A53TS87N). Aggregations monitored in vitro by fluorescent dye thioflavin T were only present in A53T mutants after four days, further suggesting A53TS87N mutants more closely adopt wild-type properties. Similarily, a ten-day assay utilizing amyloid-binding dye congo red showed lower aggregation for A53TS87N that A53T and even wild-type protein. Further experimentation will help confirm if the S87N mutation prevents PD symptoms in cells with A53T AS. Clinical implications include gene and protein therapies for PD patients.
School:
University of Illinois at Urbana-Champaign
Department:
Molecular & Integrative Physiology
Research Advisor:
Julia M. George
Department of Research Advisor:
Molecular and Integrative Physiology
Year of Publication:
2006