Exploiting Post-mitotic Yeast Cultures to Model Neurodegeneration

Abstract

Over the last few decades, the budding yeast Saccharomyces cerevisiae has been extensively used as a valuable organism to explore mechanisms of aging and human age-associated neurodegenerative disorders. Yeast models can be used to study loss of function of disease-related conserved genes and to investigate gain of function activities, frequently proteotoxicity, exerted by non-conserved human mutant proteins responsible for neurodegeneration. Most published models of proteotoxicity have used rapidly dividing cells and suffer from a high level of protein expression resulting in acute growth arrest or cell death. This contrasts with the slow development of neurodegenerative proteotoxicity during aging and the characteristic post-mitotic state of the affected cell type, the neuron. Here, we will review the efforts to create and characterize yeast models of neurodegeneration using the chronological life span model of aging, and the specific information they can provide regarding the chronology of physiological events leading to neurotoxic proteotoxicity-induced cell death and the identification of new pathways involved.

Keywords: Saccharomyces cerevisiae; chronological life span; inducible yeast model; mitochondria; neurodegenerative disorder; proteotoxicity; reactive oxygen species.

FIGURE 1

Scheme depicting the strategic planning for the creation of yeast models of neurodegenerative disorders. The strategies used for the construction of yeast models of human monogenic neurodegenerative diseases depend on genetic and pathophysiological constraints. Whether the disease is dominant or recessive, whether the phenotype results from a gain or a loss of function of the protein involved, and whether the gene is functionally conserved or not from yeast to humans are determinants of the kind of yeast model that can be generated. References located in the relevant text boxes provide actual examples in the literature of yeast models of neurodegenerative disorders. See full explanation in the text. Figure modified from Fontanesi et al. (2009).

FIGURE 2

The chronological life span (CLS) assay. In a typical CLS assay, yeast strains from frozen stocks (–80°C) are patched onto YPD agar plates (2% glucose) and incubated at 30°C. The following day, cells are inoculated into 10 ml of SDC media and grown overnight. After 24 h, cells are inoculated into 50 ml of synthetic (SDC) media in 250-ml flasks to an optical density at 600 nm (OD₆₀₀) of 0.250. Cultures are then grown with shaking (250 rpm) at 30°C. We recommend that all flasks are capped using Bio-Silico plugs that ensure sterility and maximize airflow (Hirschmann, Louisville, KY, United States). Maximum cell density is normally reached after 48 h of growth in SDC, therefore we consider 3 days after inoculation as Day 0 of CLS. Subsequently, cellular viability is determined every other day by either a clonogenic approach using the colony formation unit (CFU) assay of propidium iodide staining and flow cytometry analyses (PI-FCM) as described (Ocampo and Barrientos, 2012; Ocampo et al., 2012). The data obtained from viability analysis is then used to construct survival curves.

FIGURE 3

Scheme of some of the experiments used to monitor mitochondrial damage during CLS. During CLS of wild-type yeast cultures expressing or not the toxic proteins are collected at different time points and analyzed for markers of mitochondrial function including: (1) The index of respiratory competence (Parrella and Longo, 2008) or proportion of cells that are able to form colonies in media containing respiratory (glycerol) versus fermentable (glucose) carbon sources. (2) Endogenous cell respiration measured polarographically and mitochondrial respiratory chain (MRC) enzymatic activities measured spectrophotometrically (Barrientos et al., 2009). (3) Mitochondrial membrane potential estimated in vivo by flow cytometry using the fluorescent probe tetramethylrhodamine ethyl ester (TMRE) (Ocampo et al., 2010). (4) Oxidative stress and damage: mitochondrial and cellular reactive oxygen species (ROS) production can be measured in whole cells by using the oxidant-sensitive fluorescent probes, MitoSOX Red and dihydroethidium (DHE) as described (Ocampo et al., 2012). Additionally, resistance to oxidative stress can be determined by exposing cells to high H₂O₂ concentrations (up to 100 mM) for 1 h prior to testing growth in complete solid media. Also, mtDNA point mutation frequency can be estimated by counting the proportion of cells that acquire resistance to specific antibiotics (chloramphenicol or erythromycin). Cells are plated in respiratory media supplemented with the drugs as described (O’Rourke et al., 2002).

FIGURE 4

β-Estradiol inducible yeast models of polyglutamine disorders. (A) Chronology of polyQ-GFP protein accumulation, followed by fluorescence microscopy, in cells induced with 50 nM β-estradiol. The bar is 5 μm. (B,C) Yeast CLS. Survival of wild-type cells expressing 25Q or 103Q from a β-estradiol-inducible promoter activated with the indicated amounts of inducer or supplemented with the solvent (ethanol) was estimated by propidium iodide (PI) staining and flow cytometry analysis of 10,000 cells. Data are average of three samples in % of cells alive at day 0. In (C) a β-estradiol titration was performed. (D) Effect of increased mitochondrial biogenesis by HAP4 overexpression on CLS of yeast expressing 103Q from day 0 in the stationary phase. Error bars represent SEM for three independent experiments. (E) Effect of growth in synthetic medium containing ethanol and glycerol as non-fermentable (respiratory) carbon sources (WOEG) on 103Q yeast CLS compared to synthetic medium containing glucose as fermentable carbon source (WOGLU). SD <1 for all samples, n = 3. (F) Effect of calorie restriction (CR) modeled by growing the cells in the presence of 0.5% glucose vs. non-CR (2% glucose) in 103Q yeast CLS. SD <1 for all samples, n = 3. This figure was constructed using panels previously published in Ruetenik et al. (2016) with permission since they were published under the terms of the Creative Commons Attribution (CC BY) license.