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Yeast Hub Lab

Research in the Yeast Hub Lab explores the molecular mechanisms underlying neurodegenerative diseases (ND). We use  humanized yeast cells as  a model system and adapt methods ranging from bioluminescence and confocal microscopy to gene manipulation strategies. Our current focus is on models for disorders related to α-synuclein and tau. Our lab collaborates closely with the Yeast Biotechnology Group at the KUL run by Joris Winderickx.

In order to study the cellular and molecular mechanisms underlying ND, different model systems are used including mammalian (mainly mice) and non-mammalian species (Drosophila melanogaster, Caenorhabditis elegans, zebrafish). Recently, also the budding yeast Saccharomyces cerevisiae (S. cerevisiae) has manifested itself as a valuable model to provide further insights into the mechanisms of human diseases and in many occasions molecular biology of yeast has led to discovery of proteins and cellular pathways that are of relevance for human diseases. If the gene implicated in the human disease is conserved in yeast, its (mal)function can be directly studied in this organism. If the gene has no yeast orthologue, heterologous expression may provide important clues about a possible (mal)function of the human protein. Furthermore, the yeast cell system presents some major advantages over other cell models. First, S. cerevisiae is well defined genetically as the genome was fully sequenced in 1997. Second, yeast cells display a high degree of conservation of cellular processes and molecular pathways (including cell cycle, cell death and survival, vesicular trafficking, …) with human cells; about 60% of the yeast genes show sequence homology to a human orthologue; and 30% of known genes involved in human disease have yeast homologs. Finally,  genetic manipulations are easy, low-cost and require short generation time. 

Yeast calcium homeostasis

As in mammalian cells, yeast intracellular calcium signaling is crucial for a myriad of biological processes ranging from cell survival to cell death. Yeast cells also bear homologs of the major components of the calcium signaling toolkit in mammalian cells, including channels, co-transporters and pumps.  We detect changes of intracellular calcium concentration in yeast cells transfected with apoaequorin. When reconstituted with coelenterazine, aequorin emits light upon binding free calcium and acts as a bioluminescent indicator for calcium. Using yeast single- and multiple-gene deletion strains of various plasma membrane and organellar calcium transporters, combined with manipulations to estimate intracellular calcium storage, we evaluate the contribution of individual transport systems to intracellular calcium homeostasis1.

Yeast calcium homeostasis. Recordings of cytosolic calcium ([Ca2+]in) as a function of time during application of 10 mM external Ca2+. Following the Ca2+ pulse, 80 mM glucose was re-added which produced a transient elevation of cytosolic calcium. Thereafter, cells were briefly exposed to Ca2+ free intracellular medium prior to membrane permeabilization using Triton X-100. Permeabilization produced a two phase calcium release signal. Green trace denotes the first derivative of the calcium release signal.

Calcium homeostasis in S. cerevisiae. Extracellular calcium enters the cytosol through Cch1/Mid1 channel complex and an unknown transporter (boxed question mark). Resting cytosolic calcium is finely tuned by vacuolar calcium uptake trough Vcx1 and Pmc1 and by calcium uptake in the ER/Golgi trough Pmr1 and Cod1.

Toxicity of a-synuclein in yeast

Parkinson's disease (PD) is the second most common neurodegenerative disorder in the Western world. The histopathological hallmark of PD is the loss of dopaminergic neurons from the substantia nigra associated with the accumulation of misfolded a-synuclein in the form of Lewy bodies. Most cases of PD are sporadic but about 10-15% are familial and several susceptibility genes have been described. α-Synuclein was the first gene that was found to play a role in the pathogenesis of PD. Mutations A53T, A30P, and E46 K, duplications and triplications of this gene have been shown to result in parkinsonism. In addition, aggregated α-synuclein has also been identified as the major component of Lewy bodies in the brains of sporadic PD patients. These findings emphasize α-synuclein as a major target in the research on the molecular mechanism underlying PD. We use S. cerevisiae as a model to study α-synuclein-linked pathology in PD. Yeast cells, which do not express an ortholog of α-synuclein, are transformed with a plasmid containing human α-synuclein.

Spotting assays and growth curve analysis reveal α-synuclein-induced growth retardation2.  Since α-synuclein may form calcium permeable pores in the plasma membrane and calcium stress could be a determinant of the selective vulnerability of dopaminergic neurons in PD, we also look at the impact of α-synuclein on yeast calcium homeostasis. Expression of α-synuclein in wild-type yeast results in elevated glucose-indced calcium responses2 and resting calcium levels. The selective deletion of key players in yeast calcium homeostasis, including vacuolar and plasma membrane calcium transporters did not protect cells from α-synuclein toxicity. However, deletion of the Golgi Ca2+/Mn2+-ATPase Pmr1, the orthologue of human SPCA1, largely suppressed α-synuclein-induced effects on cell growth and calcium homeostasis. Therefore, these results uncovered that Pmr1 is a phylogenetically conserved mediator of α-synuclein-driven changes in calcium homeostasis.

Based on these findings, we hypothesize that defects in vesicular trafficking underlie α-synuclein- induced calcium dysregulation. Calcium efflux across the plasma membrane in yeast is thought to mainly rely on exocytosis of ER/Golgi-derived secretory vesicles. Thus, the enhanced glucose-induced calcium responses in α-synuclein expressing cells may reflect diminished calcium efflux caused by α-synuclein-induced disruption of exocytosis or reduction of the vesicular pool at the plasma membrane. To establish this link, we currently exploring calcium homeostasis in yeast mutants affected in the secretory pathway. 

α-Synuclein retards growth and enhances glucose-induced calcium responses in yeasts cells.  Spotting growth assay of wild-type yeast transformed with empty vector (right) or α-synuclein (left). Optical density 600 nm is used to evaluate growth in wild-type yeast transformed with empty vector (black) or α-synuclein (red). Confocal fluorescence microscopic analysis of wild type cells expressing GFP or GFP-tagged α-synuclein and Western Blot analysis of α-synuclein expression in wild type yeast cells. Glucose-induced calcium responses in wild-type cells transformed with empty vector (black) or α-synuclein (red). At times indicated 10 mM Ca2+ was first added followed by 80 mM glucose. ∆Ca2+ reflects the difference in peak amplitude of glucose-induced calcium responses between cells transformed with empty vector and α-synuclein

1P D'hooge, C Coun, V Van Eyck, L Faes, R Ghillebert, L Mariën, J Winderickx and G Callewaert (2015). Ca2+ homeostasis in the budding yeast Saccharomyces cerevisiae: Impact of ER/Golgi Ca2+ storage. Cell Calcium  58(2), 226-235. pubmed

2S Buttner,L Faes, WN Reichelt, F Broeskamp, L Habernig,S Benke, N Kourtis, D Ruli, D Carmona-Gutierrez, T Eisenberg, P D'Hooge, R Ghillebert, V Franssens, A Harger, TR Pieber, P Freudenberger, G Kroemer, SJ Sigrist, J Winderickx, G Callewaert, N Tavernarakis and F Madeo (2013). The Ca2+/Mn2+ ion-pump PMR1 links elevation of cytosolic Ca2+ levels to a-synuclein toxicity in Parkinson's disease models. Cell Death and Differentiation 20, 465–477. pubmed