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A Method to Study α-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model
Published on: November 25, 2022
alpha-Synuclein fission yeast model: concentration-dependent aggregation without plasma membrane localization or
Katrina A Brandis1, Isaac F Holmes, Samantha J England
1Biology Department, Lake Forest College, Lake Forest, IL 60045, USA.
Abstract:
Despite fission yeast's history of modeling salient cellular processes, it has not yet been used to model human neurodegeneration-linked protein misfolding. Because alpha-synuclein misfolding and aggregation are linked to Parkinson's disease (PD), here, we report a fission yeast (Schizosaccharomyces pombe) model that evaluates alpha-synuclein misfolding, aggregation, and toxicity and compare these properties with those recently characterized in budding yeast (Saccharomyces cerevisiae). Wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T) were expressed with thiamine-repressible promoters (using vectors of increasing promoter strength: pNMT81, pNMT41, and pNMT1) to test directly in living cells the nucleation polymerization hypothesis for alpha-synuclein misfolding and aggregation. In support of the hypothesis, wild-type and A53T alpha-synuclein formed prominent intracellular cytoplasmic inclusions within fission yeast cells in a concentration- and time-dependent manner, whereas A30P and A30P/A53T remained diffuse throughout the cytoplasm. A53T alpha-synuclein formed aggregates faster than wild-type alpha-synuclein and at a lower alpha-synuclein concentration. Unexpectedly, unlike in budding yeast, wild-type and A53T alpha-synuclein did not target to the plasma membrane in fission yeast, not even at low alpha-synuclein concentrations or as a precursor step to forming aggregates. Despite alpha-synuclein's extensive aggregation, it was surprisingly nontoxic to fission yeast. Future genetic dissection might yield molecular insight into this protection against toxicity. We speculate that alpha-synuclein toxicity might be linked to its membrane binding capacity. To conclude, S. pombe and S. cerevisiae model similar yet distinct aspects of alpha-synuclein biology, and both organisms shed insight into alpha-synuclein's role in PD pathogenesis.
Insights
Fission yeast models alpha-synuclein misfolding and aggregation, key to Parkinson's disease (PD). Unexpectedly, alpha-synuclein aggregated without toxicity in fission yeast, unlike in budding yeast.
Area of Science:
- Cell Biology
- Neuroscience
- Biochemistry
Background:
- Alpha-synuclein misfolding and aggregation are central to Parkinson's disease pathogenesis.
- Fission yeast (Schizosaccharomyces pombe) has been underutilized for modeling protein misfolding diseases.
- Budding yeast (Saccharomyces cerevisiae) offers a model for alpha-synuclein aggregation, but differences exist.
Purpose of the Study:
- To establish and characterize a fission yeast model for alpha-synuclein misfolding, aggregation, and toxicity.
- To compare alpha-synuclein behavior in fission yeast versus budding yeast.
- To investigate the nucleation-polymerization hypothesis for alpha-synuclein aggregation.
Main Methods:
- Expression of wild-type and mutant alpha-synuclein (A30P, A53T, A30P/A53T) in fission yeast using inducible promoters.
- Quantitative assessment of alpha-synuclein aggregation and localization within fission yeast cells.
- Comparison of aggregation kinetics, localization, and toxicity with existing budding yeast models.
Main Results:
- Wild-type and A53T alpha-synuclein formed intracellular cytoplasmic inclusions in fission yeast in a dose- and time-dependent manner.
- A30P and A30P/A53T mutants remained diffuse, supporting the nucleation-polymerization hypothesis.
- Unlike in budding yeast, alpha-synuclein did not localize to the plasma membrane in fission yeast, and aggregation was non-toxic.
Conclusions:
- Fission yeast provides a valuable model for studying alpha-synuclein aggregation, revealing distinct behaviors compared to budding yeast.
- The lack of plasma membrane targeting and toxicity in fission yeast offers new avenues for understanding Parkinson's disease mechanisms.
- Both fission and budding yeast models contribute complementary insights into alpha-synuclein's role in Parkinson's disease.
