Lake Forest College, Lake Forest, IL 60045
Parkinson disease (PD) is an incurable neurodegenerative disease that affects over one million Americans and results from the selective loss of dopaminergic neurons in the substantia nigra. The misfolding and aggregation of the protein alpha-synuclein is the likely cause of cell death in PD. A popular hypothesis is that increasing the degradation of alpha-synuclein may protect the cell from its toxicity and aggregation. While the lysosome is pharmacologically implicated in the degradation of alpha-synuclein, the genetic link between PD and autophagy is less clear. A major route for delivering proteins to the lysosome is macroautophagy (hereafter referred to as autophagy). Autophagy is a highly conserved catabolic process in eukaryotes used to recycle and/or catabolize damaged or excess protein and organelles. We hypothesized that autophagy protects cells from alpha- synuclein toxicity, and tested it by genetically inhibiting the expansion or nucleation steps within autophagy in an alpha-synuclein budding yeast model. We asked if alpha-synuclein would accumulate, relocalize and be more toxic in strains knocked out for autophagy genes (Atg) essential to those key steps. Thus far, in atg17Δ (needed for expansion), and atg2Δ and atg18Δ (needed for nucleation), the cellular localization of alpha-synuclein was subtly altered, but none induced toxicity. In atg2Δ yeast, alpha-synuclein aggregated more extensively while still maintaining its plasma membrane localization. While, in atg17Δ yeast, alpha-synuclein became more cytoplasmically diffuse than plasma membrane localized. In these autophagy-deficient strains, alpha-synuclein did accumulate, but to varying extents. However, the alpha-synuclein constructs previously used by our lab had an erroneous 140th amino acid (a glycine in lieu of an alanine) due to incorrect PCR primer design during subcloning. Subsequently, we corrected this sequence error and we are currently comparing the properties of the corrected alpha-synuclein constructs with the mutant ones previously examined. (Supported by APDA, NSF-MRI, NSF- CCLI & NIH R15)
[Abstract (DOC)]