Parkinson's disease (PD) is a common neurodegenerative disorder that results from the selective loss of midbrain dopaminergic neurons. Misfolding and aggregation of the protein alpha-synuclein, oxidative damage, and proteasomal impairment are all hypotheses for the molecular cause of this selective neurotoxicity. Here, we describe a Saccharomyces cerevisiae model to evaluate the misfolding, aggregation, and toxicity-inducing ability of wild-type alpha-synuclein and three mutants (A30P, A53T, and A30P/A53T), and we compare regulation of these properties by dysfunctional proteasomes and by oxidative stress. We found prominent localization of wild-type and A53T alpha-synuclein near the plasma membrane, supporting known in vitro lipid-binding ability. In contrast, A30P was mostly cytoplasmic, whereas A30P/A53T displayed both types of fluorescence. Surprisingly, alpha-synuclein was not toxic to several yeast strains tested. When yeast mutants for the proteasomal barrel (doa3-1) were evaluated, delayed alpha-synuclein synthesis and membrane association were observed; yeast mutant for the proteasomal cap (sen3-1) exhibited increased accumulation and aggregation of alpha-synuclein. Both sen3-1and doa3-1 mutants exhibited synthetic lethality with alpha-synuclein. When yeasts were challenged with an oxidant (hydrogen peroxide), alpha-synuclein was extremely lethal to cells that lacked manganese superoxide dismutase Mn-SOD (sod2Delta) but not to cells that lacked copper, zinc superoxide dismutase Cu,Zn-SOD (sod1Delta). Despite the toxicity, sod2Delta cells never displayed intracellular aggregates of alpha-synuclein. We suggest that the toxic alpha-synuclein species in yeast are smaller than the visible aggregates, and toxicity might involve alpha-synuclein membrane association. Thus, yeasts have emerged effective organisms for characterizing factors and mechanisms that regulate alpha-synuclein toxicity.
In Parkinson's disease (PD), misfolded and aggregated α-synuclein protein accumulates in degenerating midbrain dopaminergic neurons. The amino acid alanine-76 in α-synuclein and phosphorylation at serine-87 and serine-129 are thought to regulate its aggregation and toxicity. However, their exact contributions to α-synuclein membrane association are less clear. We found that α-synuclein is indeed phosphorylated in fission yeast and budding yeast, the two models that we employed for assessing α-synuclein aggregation and membrane association properties, respectively. Surprisingly, blocking serine phosphorylation (S87A, S129A, and S87A/S129A) or mimicking it (S87D, S129D) altered α-synuclein aggregation in fission yeast. Either blocking or mimicking this phosphorylation increased endomembrane association in fission yeast, but only mimicking it decreased plasma membrane association in budding yeast. Polar substitution mutations of alanine-76 (A76E and A76R) decreased α-synuclein membrane association in budding yeast and decreased aggregation in fission yeast. These yeast studies extend our understanding of serine phosphorylation and alanine-76 contributions to α-synuclein aggregation and are the first to detail their impact on α-synuclein's plasma membrane and endomembrane association.
In Parkinson's disease (PD), midbrain dopaminergic neuronal death is linked to the accumulation of aggregated α-synuclein. The familial PD mutant form of α-synuclein, E46K, has not been thoroughly evaluated yet in an organismal model system. Here, we report that E46K resembled wild-type (WT) α-synuclein in Saccharomyces cerevisiae in that it predominantly localized to the plasma membrane, and it did not induce significant toxicity or accumulation. In contrast, in Schizosaccharomyces pombe, E46K did not associate with the plasma membrane. Instead, in one strain, it extensively aggregated in the cytoplasm and was as toxic as WT. Remarkably, in another strain, E46K extensively associated with the endomembrane system and was more toxic than WT. Our studies recapitulate and extend aggregation and phospholipid membrane association properties of E46K previously observed in vitro and cell culture. Furthermore, it supports the notion that E46K generates toxicity partly due to increased association with endomembrane systems within cells.
Eukaryotic cells use a series of membrane transporters to control the movement of lipids across their plasma membrane. Several tools and techniques have been developed to analyze the activity of these transporters in the plasma membrane of mammalian cells. Among them, assays based on fluorescence microscopy in combination with fluorescent lipid probes are particularly suitable, allowing visualization of lipid internalization in living cells. Here, we provide a step-by-step protocol for mammalian cell culture, lipid probe preparation, cell labeling, and confocal imaging to monitor lipid internalization by lipid flippases at the plasma membrane based on lipid probes carrying a fluorophore at a short-chain fatty acid. The protocol allows studying a wide range of mammalian cell lines, to test the impact of gene knockouts on lipid internalization at the plasma membrane and changes in lipid uptake during cell differentiation. Key features Visualization and quantification of lipid internalization by lipid flippases at the plasma membrane based on confocal microscopy. Assay is performed on living adherent mammalian cells in culture. The protocol can be easily modified to a wide variety of mammalian cell lines.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.