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Frontiers in Molecular Neuroscience
Modeling Neurodegeneration in Yeast View all 14 Articles
Ralf J. Braun
Danube Private University, Austria
University of Connecticut Health Center, United States
Markus Babst
The University of Utah, United States
Open Supplemental Data
Front. Mol. Neurosci., 05 November 2018 | https://doi.org/10.3389/fnmol.2018.00406
The Impact of ESCRT on Aβ1-42 Induced Membrane Lesions in a Yeast Model for Alzheimer's Disease
Gernot Fruhmann1†, Christelle Marchal2†‡, Hélène Vignaud2, Mathias Verduyckt1, Nicolas Talarek3, Claudio De Virgilio4, Joris Winderickx1* and Christophe Cullin2*
1Functional Biology, KU Leuven, Leuven, Belgium
2Institut de Chimie et Biologie des Membranes et des Nano-objets, University of Bordeaux, CNRS UMR 5248, Pessac, France
3Institut de Génétique Moléculaire de Montpellier, University of Montpellier, CNRS, Montpellier, France
4Department of Biology, Université de Fribourg, Fribourg, Switzerland
Aβ metabolism plays a pivotal role in Alzheimer's disease. Here, we used a yeast model to monitor Aβ42 toxicity when entering the secretory pathway and demonstrate that processing in, and exit from the endoplasmic reticulum (ER) is required to unleash the full Aβ42 toxic potential. Consistent with previously reported data, our data suggests that Aβ42 interacts with mitochondria, thereby enhancing formation of reactive oxygen species and eventually leading to cell demise. We used our model to search for genes that modulate this deleterious effect, either by reducing or enhancing Aβ42 toxicity, based on screening of the yeast knockout collection. This revealed a reduced Aβ42 toxicity not only in strains hampered in ER-Golgi traffic and mitochondrial functioning but also in strains lacking genes connected to the cell cycle and the DNA replication stress response. On the other hand, increased Aβ42 toxicity was observed in strains affected in the actin cytoskeleton organization, endocytosis and the formation of multivesicular bodies, including key factors of the ESCRT machinery. Since the latter was shown to be required for the repair of membrane lesions in mammalian systems, we studied this aspect in more detail in our yeast model. Our data demonstrated that Aβ42 heavily disturbed the plasma membrane integrity in a strain lacking the ESCRT-III accessory factor Bro1, a phenotype that came along with a severe growth defect and enhanced loading of lipid droplets. Thus, it appears that also in yeast ESCRT is required for membrane repair, thereby counteracting one of the deleterious effects induced by the expression of Aβ42. Combined, our studies once more validated the use of yeast as a model to investigate fundamental mechanisms underlying the etiology of neurodegenerative disorders.
Amyloid β1-42 (referred to as Aβ42) is an intensively investigated peptide involved in Alzheimer's disease (AD) but beyond its role as a possibly malignant factor, the physiological functions of Aβ42 are still speculative. It has been shown that it has a role in activity dependent synaptic vesicle release and there is evidence that it might act as an antimicrobial agent in the brain, but besides that, not much is known about its physiological function (Pearson and Peers, 2006;Abramov et al., 2009; Kagan et al., 2012; Kumar et al., 2016). Thus, although several efforts have been put in investigating its role in AD, still most of its functions remain elusive. In humans, the membrane bound amyloid precursor protein, APP, is processed in two possible ways: it can enter the non-amyloidogenic pathway or it might be cleaved by β- and γ-secretases in the amyloidogenic pathway thus producing Aβ peptides of lengths between 37 and 43 amino acids (LaFerla et al., 2007). In AD, these peptides are then secreted and form inert extracellular plaques, but monomers and especially small oligomers are transported to several intracellular organelles where they reveal their toxic potential. For instance, strong evidence was found for that Aβ42 perturbs proper function of mitochondria through blocking respiration at complex IV, resulting in cells producing more reactive oxygen species (ROS) and eventually apoptotic cell death (Wang et al., 2010; Ittner and Gotz, 2011; Benilova et al., 2012; Jackrel et al., 2014; Alexandrov et al., 2016; Vicente Miranda et al., 2016).
In the past decade, yeast was established as a model organism for studying fundamental aspects related to neurodegenerative disorders such as Huntington's, Parkinson's, or AD (Winderickx et al., 2008; Franssens et al., 2010; Swinnen et al., 2011; Porzoor and Macreadie, 2013; Jiang et al., 2016; Verduyckt et al., 2016; Fruhmann et al., 2017). Since most of the overall cellular architecture and most basic biochemical processes are conserved between mammalian cells and yeast, and given the fact that about 20–30 per cent of all human genes have orthologs in Saccharomyces cerevisiae (Foury, 1997; Hamza et al., 2015; Liu et al., 2017), it is obvious that certain disease mechanisms can be studied in this easy to handle model organism. In connection to AD and Aβ42, we and others reported that expression of this peptide triggers toxicity in yeast when targeted to the secretory pathway as to mimic its multi-compartment trafficking observed in mammalian systems. This led to the observation that Aβ42 expression in yeast alters endocytosis of plasma membrane resident proteins (Treusch et al., 2011; D'Angelo et al., 2013), induces ER-stress and the unfolded protein response (Chen et al., 2017) and triggers mitochondrial dysfunction (D'Angelo et al., 2013; Chen and Petranovic, 2015; Chen et al., 2017). In the studies presented in this paper, we used previously reported constructs (D'Angelo et al., 2013; Vignaud et al., 2013) where the yeast mating type α-prepro factor directs Aβ42 into the Golgi. Here, the α-prepro factor is cleaved off, followed by transport of the peptide to the plasma membrane. In addition, the constructs contain a C-terminal linker-GFP tag in order to ensure stable expression and easy localization of Aβ42 in the yeast cells. Besides the wild-type Aβ42 and the clinical arctic mutant, we also expressed two synthetic mutants generated by random mutagenesis and previously shown to be either more toxic (Aβ42G37C) to, or to be moderately toxic (Aβ42L34T) compared to Aβ42wt (D'Angelo et al., 2013; Vignaud et al., 2013). Using these constructs, we performed genome-wide screenings as to identify Aβ42 toxicity modulators. We confirm the previously reported Aβ42 toxicity phenotypes and in addition demonstrate that Aβ42 introduces membrane lesions that require the ESCRT system in order to become repaired.
Yeast Strains, Plasmids, and Media
We used the haploid Saccharomyces cerevisiae strain BY4741 MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 and BY4742 MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 for all specified experiments. All deletion strains were obtained from the commercial EUROSCARF knock-out library (Y.K.O. collection). For a full list of strains used in this study see Table 1.
TABLE 1. Yeast strains used in this study.
The pYe plasmids with the galactose inducible, episomal αAβ42-linker-GFP isoforms, the similar Aβ42-GFP construct without the α-prepro sequence and the control constructs for expressing of either GFP (ev-GFP) or a α-prepro-GFP fusion (ev-αGFP) were described previously (Tong et al., 2001). The p426-GAL vector (Addgene) was used as additional empty vector (ev) control. For a full list of plasmids used in this study see Table 2.
TABLE 2. Plasmids used in this study.
Standard yeast techniques were applied. We used minimal medium containing Yeast Nitrogen Base (YNB) without ammonium sulfate (FORMEDIUM). Supplements were ammonium sulfate (5 g/L; VWR), histidine (100 mg/L; MP Biomedicals), methionine (20 mg/L; Acros Organics), leucine (30 mg/L; FORMEDIUM) and lysine (30 mg/L; FORMEDIUM). Synthetic Drop-Out (SD) medium was used for microscopy and contained YNB with ammonium sulfate and was depleted for either uracil or uracil and leucine (FORMEDIUM). For solid media, 1.5% Difco-agar (BD) was added. Pre-cultures were grown on medium supplemented with 4% glucose and gene expression was induced by washing the cells with medium without sugar followed by transfer to medium supplemented with 2% galactose.
Synthetic Genetic Array and Suppressor screening
The synthetic genetic array (SGA) screening was essentially performed as previously described in Tong et al. (2001). The query strain (MATα can1Δ::STEpr-HIS5sp lyp1Δ his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 LYS2) expressing αAβ42wt-linker-GFP (further designated as αAβ42wt), the αAβ42G37C-linker-GFP (designated αAβ42G37C) or the ev-αGFP was mated with the non-essential deletion mutant array (MATa target_gene::kanMX4 his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) on SD plates lacking uracil. Diploids were selected on SD medium lacking uracil but containing G418 (geneticin). Next, sporulation was induced by plating diploids onto sporulation medium containing G418. Then, haploids were selected in two steps. First, spores were plated onto YNB lacking arginine, lysine, and histidine but containing canavanine and thialysine, which ensures uptake of canavanine. This allowed growth of MATa haploids only. In a second step, the selected haploids were grown on YNB lacking uracil, arginine, lysine, and histidine but containing canavanine, thialysine and G418 to select for haploid knock-out mutants still carrying the αAβ42wt, αAβ42G37C, or ev-αGFP plasmids. Growth analysis was performed with the ScreenMill software (Dittmar et al., 2010).
In a second so-called suppressor screening, the Euroscarf collection of deletion strains was pooled transformed with αAβ42G37C. Transformants were plated on minimal medium lacking uracil. After incubation, transformants were selected that grew similar as the isogenic wild-type carrying the empty vector control, also when replica plated on SD medium supplemented with casamino acids. Transformants were then used for bar-code PCR sequencing as to identify their corresponding ORF deletion.
Electron microscopic analysis of yeast cells was done similar as previously described (Lefebvre-Legendre et al., 2005). Briefly, pellets of yeast cells were placed on the surface of a copper EM grid (400 mesh) coated with formvar. Grids were immersed in liquid propane held at -180°C by liquid nitrogen and then transferred in a 4% osmium tetroxide solution in dry acetone at -82°C for 72 h. They were warmed progressively to RT, and washed three times with dry acetone and stained with 1% uranyl acetate. After washing in dry acetone, the grids were infiltrated with araldite (Fluka). Ultra-thin sections were contrasted with lead citrate and observed in an electron microscope (80 kV; 7650; Hitachi) at the EM facility of the Bordeaux Imaging Center.
Growth Profile Analysis and Spot Assays
Cells were grown under non-inducing conditions in 96-well plates shaking at 30°C in a Multiskan GO or Multiskan FC microplate spectrophotometer (Thermo Scientific) to an OD595 nm or OD600 nm, respectively, of 0.5 to 0.9. Cells were washed with minimal medium containing galactose and diluted to an OD595 nm or OD600 nm, respectively, of 0.5 after which growth was monitored every 2 h by OD measurement. Four different transformants were taken per experiment and at least three independent experiments were performed. Growth curves were analyzed with GraphPad Prism v7.03, error bars represent standard deviations.
For spot assays, serial dilutions of precultures were spotted on solid medium containing either glucose or galactose and cells were grown at 30°C. The plates were scanned at days 3 to 6.
Cytometry and Fluorescence Microscopy
To monitor plasma membrane disruption with propidium iodide (PI) staining and the formation of ROS by dihydroethidium (DHE) staining, cells were grown in SD medium lacking uracil and containing 4% glucose. Once in exponential phase, the cells were washed with and re-suspended in SD medium without uracil but containing 2% galactose as to induce expression of αAβ42-linker-GFP and control constructs. Flow cytometric analysis was performed on at least four independent transformants with a Guava easyCyte 8HT benchtop flow cytometer (Millipore) after staining with 5 μM PI for 30 min at 30°C and subsequent washing. Data were analyzed with FlowJo v10 and GraphPad Prism 7.03 software packages. Gates were set in FlowJo v10 with single stained ev-αGFP samples. Further statistical analysis was done in GraphPad Prism v7.03. Error bars represent standard deviations and asterisks the significance calculated with an ordinary Two-way ANOVA.
For the FM4-64/CMAC (Thermo Fisher) and Nile Red (Acros) stainings, the cells were grown as described above. After pre-incubating the cells with CAMAC at 30°C for 30 min, FM4-64 stainings were performed as described before (Zabrocki et al., 2005). For the Nile Red staining, the cells were fixed with formaldehyde (4% final concentration) and stained with 2% Nile Red (60 μg/mL stock) for 30 min with shaking at 30°C. Then, the cells were washed twice with PBS and either stored at 4°C or taken immediately for fluorescence microscopy.
For epifluorescence pictures, cells were pre-grown in selective glucose (4%) containing SD medium to exponential phase. After transfer to SD medium containing 2% galactose to induce expression of αAβ42-linker-GFP, the cells were grown at 30°C and pictures were taken after different time intervals using a Leica DM4000B or a DMi8 microscope. For Hoechst stainings, the cells were incubated with 20 nM Hoechst 33342 (Sigma-Aldrich) for 10 min. Pictures were deconvolved with Huygens Essential software (v18.04.0p4 64, Scientific Volume Imaging B.V.) and further processed with the standard FIJI software package (v1.51n) (Schindelin et al., 2012).
Immunological Techniques
Cultures were grown to exponential phase in 4% glucose containing SD medium, transferred to 2% galactose containing medium and grown overnight. Then, three OD units were harvested by centrifugation and protein extracts were prepared by using an alkaline lysis method. The cells were permeabilized with 0.185 M NaOH plus 2% β-mercaptoethanol. After 10 min incubation on ice, trichloroacetic acid (TCA) was added to a final concentration of 5%, followed by an additional 10 min incubation step on ice. Precipitates were collected by centrifugation for 5 min at 13000 g and pellets were resuspended in 50 μL of sample buffer (4% sodium dodecyl sulfate, 0.1 M Tris-HCl pH 6.8, 4 mM EDTA, 20% glycerol, 2% β-mercaptoethanol, and 0.02% bromophenol blue) plus 25 μL of 1 M Tris-Base. Samples were separated by standard SDS-PAGE on 12% polyacrylamide gels and further analyzed using standard Western blotting techniques. An anti-GFP primary antibody (Sigma-Aldrich) and anti-Mouse (GAM)-HRP conjugated secondary antibody (Biorad) were used. The ECL method (SuperSignal West Pico or Femto, Thermo Scientific) was used for detection and visualization of the blots was performed with a UVP Biospectrum® Multispectral Imaging System.
αAβ42-Linker-GFP Is Toxic in an AD Yeast Model
To date, the exact molecular basis of how Aβ42 impacts cell functions remains largely elusive. To address this question, we used a yeast model transformed with plasmids carrying the galactose-inducible GAL10 promoter to control expression of wild-type or mutant Aβ42 that is N-terminally fused to the α-prepro sequence and C-terminally to a linker and GFP (in this paper referred to as αAβ42; Figure 1A) (D'Angelo et al., 2013; Vignaud et al., 2013). Besides the wild-type αAβ42 (αAβ42wt), we additionally expressed the clinical E22G arctic mutant (αAβ42arc) that is associated to a familial form of AD as well as the previously described synthetic mutants αAβ42G37C and αAβ42L34T (Figure 1A) (D'Angelo et al., 2013; Vignaud et al., 2013). We used three control vectors, i.e., an empty vector allowing for the expression of an α-prepro fused GFP (ev-αGFP), GFP alone (ev-GFP) or an empty vector just carrying the galactose promotor (ev). To disperse concerns about the processing efficacy of the α-prepro factor in the Golgi system, we tested both the BY4741 MATa and BY4742 MATα strains with all control vectors and the aforementioned wild-type and mutant αAβ42-linker-GFP constructs (Figures 1B–D and Supplementary Figure S1). Albeit the strains transformed with the ev-αGFP control grew somewhat slower than those transformed with ev-GFP or ev (Figures 1B,C and Supplementary Figure S1A), both spot assays and growth analysis in liquid medium confirmed that wild-type or mutant αAβ42 instigated a significantly higher level of toxicity that was similar in the BY4741 and the BY4742 strains (Figures 1B,C and Supplementary Figure S1). The synthetic αAβ42G37C mutant was the most toxic followed by αAβ42wt and αAβ42arc. The synthetic αAβ42L34T mutant, on the other hand, did not yield a toxic phenotype and these transformants grew similar as those expressing ev-αGFP, thereby confirming previously reported data (Vignaud et al., 2013).
FIGURE 1. Toxicity profiles of wild-type or mutant αAβ42 in the haploid Saccharomyces cerevisiae strains BY4741. (A) Schematic representation of the α-prepro-Aβ42-linker-GFP construct with indication of the mutants used in this study. (B) Spot assays under inducing (galactose) and repressing (glucose) conditions and (C) growth profiles under inducing conditions of the BY4741 wild-type strain transformed with constructs allowing for expression of αAβ42wt, αAβ42arc, αAβ42G37C, αAβ42L34T, Aβ42wt and control vectors (ev-αGFP, ev-GFP, ev) as indicated. Error bars in the growth profiles represent the standard deviation of at least four independent transformants. (D) Fluorescence microscopy pictures show localization of α-prepro fused Aβ42 mutants to the ER 6 and 24 h after induction on galactose-containing medium. 24 h after induction of gene expression the toxic αAβ42 isoforms show a more "patchy" pattern while the non-toxic αAβ42L34T still localizes at the ER. The Aβ42wt construct lacking the α-prepro sequence is seen in the cytoplasm. The scale bar represents 2 μm. (E) Growth profiles of the BY4741 wild-type strain transformed with an empty vector (ev) or constructs allowing for expression of αAβ42wt and Aβ42wt.
Fluorescence microscopy showed that all αAβ42 constructs clearly stained the perinuclear ER and to a lesser extent the cortical ER and that particularly the cells expressing the toxic αAβ42 forms often displayed ER-associated foci and filamentous structures (Figure 1D). These were previously believed to be vesicles (D'Angelo et al., 2013), but recent studies suggests that these may as well-represent ER-aggregates or clustering of ER membranes, both indicative for ER stress (Varadarajan et al., 2012, 2013; Vevea et al., 2015). In contrast, when the Aβ42wt was expressed from a construct that lacks the α-prepro sequence, the GFP fusion was mainly found to be distributed in the cytoplasm though some cells presented foci after prolonged induction (Figure 1D). Despite of these foci, the expression of the Aβ42wt construct without the α-prepro sequence only triggered a small growth retardation (Figures 1B,E). This nicely demonstrates that the processing in the ER/Golgi system is required to unleash the full toxic capacity of αAβ42.
Retention of αAβ42 in the ER Diminishes Its Toxicity
Previously, we reported that the processing of the αAβ42-linker-GFP fusion constructs in the ER/Golgi system yields three distinct isoforms when performing Western blot analysis, i.e., the α-prepro precursor (41 kDa), the glycosylated precursor (50 kDa), and the matured Aβ42-linker-GFP form (34 kDa) (D'Angelo et al., 2013; Vignaud et al., 2013). Given that the latter is shuttled into the secretory pathway, we wondered if retention of the αAβ42-linker-GFP fusion in the endoplasmic reticulum (ER) would affect its toxic capacity. To this end, we introduced a HDEL retention signal at the C-terminal end of the construct. The yeast HDEL sequence is equivalent to the mammalian KDEL retention signal that shuttles the KDEL containing proteins back to the ER lumen (Dean and Pelham, 1990; Lewis and Pelham, 1990; Ruan et al., 2017). Consistently, microscopic analysis demonstrated that while both αAβ42G37C and αAβ42G37C-HDEL are equally present at the perinuclear ER, the latter accumulated more in the peripheral cortical ER (Figure 2A). Also, Western blot analysis showed that in case of expression of αAβ42G37C-HDEL, both the non-processed αAβ42G37C precursor as well as the glycosylated version accumulated, while the fully processed Aβ42G37C was significantly reduced (Figure 2B). Next, a growth analysis was performed. This revealed that the strain expressing αAβ42G37C-HDEL construct grew much better than that with αAβ42G37C, thereby displaying a level of toxicity comparable to a strain with the αAβ42arc mutant (Figure 2C).
FIGURE 2. Retention of αAβ42 in the ER and the consequence for toxicity. (A) Fluorescence microscopy pictures showing αAβ42G37C and αAβ42G37C-HDEL localization at the perinuclear and cortical ER. Scale bar represents 2 μm. (B) Western blot analysis of total protein extracts obtained from cells expressing either αAβ42G37C and αAβ42G37C-HDEL. The different processing isoforms are indicated, i.e., αAβ42∗ indicates glycosylated αAβ42, αAβ42 indicates un-glycosylated αAβ42 and Aβ42 indicates fully processed form where the α-prepro sequence is cleaved off. Spot assays under inductive (galactose) and repressive (glucose) conditions of wild-type (C) or erv29Δ (D) cells transformed with the empty vector (ev) or constructs allowing for expression of αAβ42wt, αAβ42arc, αAβ42G37C, or αAβ42G37C-HDEL as indicated. (E) Growth profiles of erv29Δ transformed with the empty vector (ev) or constructs allowing for expression of αAβ42wt, αAβ42arc, or αAβ42G37C when grown on galactose-containing medium.
In order to confirm these results with a different approach, we expressed αAβ42wt, αAβ42arc, as well as the super-toxic αAβ42G37C mutant in a BY4741 strain deleted for ERV29, which encodes a transmembrane factor involved in COP-II dependent vesicle formation and in trafficking of the α-prepro factor from the ER to the Golgi apparatus (Belden and Barlowe, 2001). Upon deletion of ERV29, αAβ42 gets stuck in the ER and cannot transit to the Golgi. Strikingly, cytotoxicity of all tested mutants was indeed significantly diminished in this erv29Δ strain and even the super-toxic αAβ42G37C mutant almost completely lost its toxic power (Figures 2D,E). Thus, the data above confirm that αAβ42 needs to be fully processed and exit the ER in order to gain its full toxic potential.
αAβ42 Affects Mitochondrial Functioning
Since αAβ42 confers toxicity by entering the secretory pathway, we wanted to know more about the targets and processes being affected. Therefore, we first focused on mitochondria given that previous studies have associated Aβ42 to mitochondrial dysfunction in yeast (Chen and Petranovic, 2015; Chen et al., 2017; Hu et al., 2018). To this end, we co-expressed αAβ42wt, αAβ42arc, αAβ42G37C, αAβ42L34T or the ev-αGFP together with a mCherry labeled mitochondrial marker Cox4 in wild-type cells. As illustrated in Figure 3A, this suggests a toxicity dependent co-localization since αAβ42wt and even more αAβ42G37C seem to partially co-localize with mitochondria. We then performed a DHE staining to estimate the ROS levels as marker for mitochondrial dysfunction and a PI staining to monitor the amount of cells with disrupted plasma membranes as marker for cell demise (Figures 3B,C and Supplementary Figure S2). As expected, and consistent with previously reported data (Chen and Petranovic, 2015; Chen et al., 2017), also these aspects correlated to the observed αAβ42 instigated toxicity. Although our data on co-localization suggest that αAβ42 may directly interfere with mitochondrial functioning, we cannot exclude that these effects on mitochondrial function or morphology are indirect, for instance due alterations in ER-mitochondrial communication via ERMES contact sites.
FIGURE 3. αAβ42 induces mitochondrial dysfunction and cell demise. (A) Fluorescence microscopy pictures suggesting partial co-localization of toxic αAβ42 but not of the non-toxic αAβ42L34T nor of the αGFP control (green) with mitochondria (red) in wild-type cells. Hoechst staining (blue) shows mitochondrial as well as nuclear DNA. The white arrowheads indicate sites of co-localization between αAβ42 and mitochondria. Two single cells shown per strain. Scale bars represent 2 μm. Percentage of wild-type yeast cells expressing wild-type or mutant αAβ42 and a control stained with DHE as a marker for ROS-formation (B) or PI as a marker for plasma membrane integrity (C). ∗P ≤ 0.05; ∗∗P ≤ 0.01; ∗∗∗∗P ≤ 0.0001; nsP > 0.05.
Identification of Additional Processes Underlying αAβ42 Toxicity
To decipher which additional processes sustain the αAβ42 toxicity, we performed two unbiased genetic screens. In a first screening setup, we aimed to identify alleviators of Aβ42G37C toxicity. Therefore, we transformed a pooled Euroscarf knock-out (KO) library with the αAβ42G37C-linker-GFP construct and looked for transformants that grew similar as the empty vector control. Out of 90,000 transformants obtained when plated on repressive glucose containing medium, only 465 were able to form visible colonies after 72 h on galactose-containing medium where the expression of the super-toxic Aβ42 mutant is induced. Each colony was then plated on SD medium supplemented with casamino acids to manually confirm the suppressive effect on αAβ42 toxicity. Of the 465 initial clones, 268 were still able to grow. Finally, we characterized each KO strain by sequencing the PCR amplicon of its bar-code region. This led us to identify 113 different KO strains. In the second screening setup, we performed an unbiased SGA analysis with the full gene knock-out library using either αAβ42wt, the super-toxic αAβ42G37C mutant and the ev-GFP control. The additional use of the empty vector control and αAβ42wt, which displays a more moderate toxicity phenotype, allowed for more complete results since not only suppressors but also toxicity aggravators could be scored. With this SGA screen, we identified 87 additional KO strains that modulated αAβ42 toxicity, one of which contains a deletion in the overlapping genes INP52/RRT16. Finally, the 200 KO strains that were identified by either one of the screening procedures (Table 3 and Supplementary Table S1) were also transformed individually with a construct allowing for the expression of αAβ42arc, which has an intermediate toxicity similar to that of αAβ42wt and thus is again ideal to provide confirmation of both aggravators and suppressors of toxicity (D'Angelo et al., 2013). This provided an independent confirmation that all the KO strains selected in one of the two genetic screens are indeed involved in modulating αAβ42arc toxicity. For each of the KO strains, the cell density under galactose inducing conditions was monitored allowing to rank KO strains from 0 to 5 depending on the growth capacity in comparison to the wild-type strain transformed αAβ42arc. For 29 KO strains, growth was improved strongly (scored as 5), for 18 KO strains growth was clearly improved (scored as 4) and for 129 KO strains growth was only slightly better (scored as 3). On the other hand, we also identified 2 KO strains in which αAβ42arc toxicity was strongly enhanced (scored as "0") and 17 KO strains that enhanced αAβ42arc toxicity moderately (scored as 1) or weakly (scored as 2) (Table 3 and Supplementary Table S1). Next, a gene ontology (GO) analysis using the SGD Gene Ontology Slim Mapper1 allowed to sort the KO strains into functional categories depending on the gene deleted (Table 3). These included, amongst others, cytoskeleton organization and endocytosis, protein sorting and trafficking, protein ubiquitination, plasma membrane transport, cell cycle, translation, and transcription.
TABLE 3. αAβ42 toxicity modifiers.
Interestingly, among the many KO strains that alleviated αAβ42arc toxicity we found not only the strain deleted for ERV29, which is in line with the data described above, but also strains lacking other genes that impact on ER/Golgi functioning and traffic, such as the SPC2 encoded subunit of the peptidase complex, which cleaves the signal sequence from proteins targeted to the ER, EMP24, which encodes a component of the p24 complex that mediates ER-to-Golgi transport of GPI anchored proteins, or ARF2 and YPT31, which both encode GTPases required for intra-Golgi traffic. Also found were several strains lacking genes encoding mitochondrial functions and this included AIF1 that codes for the mitochondrial cell death effector, indicative that αAβ42 actively induces programmed cell death pathways. Furthermore, the fact that we retrieved the KO strains for ATG1, ATG2, and ATG20 suggests that αAβ42 may overstimulate the autophagy and the cytoplasm-to-vacuole targeting pathways (Kim and Klionsky, 2000; Nice et al., 2002). Finally, it was recently shown by transcriptome analysis that Aβ42 impacts on lipid metabolism with INO1 being most significantly upregulated (Chen et al., 2017). We found the KO strain lacking ITC1 to reduce αAβ42 toxicity. ITC1 encodes a subunit of ATP-dependent Isw2p-Itc1p chromatin remodeling complex that is required for repression of INO1. In addition, also KO strains lacking genes involved sphingolipid and ceramide metabolism were retrieved, i.e., FAA1, SUR1, YDC1. Overall, these data confirm that the noxious effect of αAβ42 is associated to changes in lipid metabolism.
The KO strains that aggravated αAβ42 toxicity were often missing functions associated to maintenance and organization of the actin cytoskeleton, endocytosis and the multivesicular body (MVB) pathway. The two strains with the strongest enhanced toxicity were those carrying a deletion of SAC6 or RVS167. Sac6 is an actin-bundling protein that is required for endocytosis (Penalver et al., 1997; Gheorghe et al., 2008). Rvs167 is a homolog of mammalian amphiphysin that interacts with actin as well and that functions in the internalization step of endocytosis (Lombardi and Riezman, 2001). As illustrated for the expression of αAβ42wt, we indeed observed a similar severe growth phenotype in both the sac6Δ and rvs167Δ mutants as compared to the isogenic wild-type strain (Figure 4A). To further confirm the link between endocytosis and αAβ42 toxicity, we also monitored the uptake of the endocytosis tracker FM4-64 by wild-type cells expressing either the toxic αAβ42wt and αAβ42G37C and non-toxic αAβ42L34T isoforms after 4 h induction on galactose-containing medium. As shown, FM4-64 already stained the vacuolar membrane within 30 min in cells expressing the non-toxic construct while no, or only a minimal staining of the vacuolar membrane was observed in cells expressing the toxic αAβ42 species, even not after 60 min of incubation (Figure 4B). This demonstrates that the latter have a direct impact on the endocytic process.
FIGURE 4. Interference of αAβ42 with endocytosis. (A) Growth profiles of wild-type cells and rvs167Δ or sac6Δ cells transformed with the empty vector (ev) or a construct allowing for expression of αAβ42wt when grown on galactose-containing medium. (B) Fluorescence microscopy pictures of strains expressing the toxic αAβ42wt and αAβ42G37C or the non-toxic αAβ42L34T stained with the endocytosis tracker FM4-64 (red) and CMAC (blue), a dye to stain the vacuolar lumen. The scale bar represents 2 μm.
αAβ42 Enhances the Occurrence of Plasma Membrane Lesions and Formation of Lipid Droplets
Closely connected to endocytosis, we noticed that our screening retrieved some ESCRT components, which function in the MVB pathway that is required for the turnover of plasma membrane proteins and lipids. The first step in MVB formation is the binding of ESCRT-0 (Hse1 and Vps27) and ESCRT-I (involving Vps28, Mvb12, Srn2, and Stp22) to ubiquitinated MVB cargoes. Next, the ESCRT-II complex (involving Vps25, Vps36, and Snf8) mediates the recruitment of ESCRT-III accessory factors (Bro1 and Doa4), which in turn loads general ESCRT-III factors (Vps20, Snf7, Did4, Chm7, Ist1, and Vps24) to direct the continued sorting of cargoes into invaginating vesicles during MVB formation. ESCRT-III dissociation factors (Vps4, Vps60, Did2, and Vta1) mediate the release and recycling of all involved factors (Hurley, 2010; Babst, 2011). All the 21 corresponding genes are not essential and could therefore be tested for their implications in αAβ42 toxicity. After transformation of the corresponding KO strains with either αAβ42wt, αAβ42G37C or the ev-αGFP control construct, we evaluated the impact of the deletions on αAβ42 toxicity through spot assays. This revealed that for several components of the MVB pathway their deletion significantly aggravated αAβ42 toxicity. This included the ESCRTIII components Did4, the ESCRTIII accessory components Bro1 and Doa4 as well as the ESCRTIII dissociation mediator Vps4 (Supplementary Figures S3, S4). Recent studies in mammalian cells have demonstrated that besides its role in the MVB pathway, ESCRT plays key roles in a variety of other processes, including membrane lesion repair (Jimenez et al., 2014; Sundquist and Ullman, 2015; Campsteijn et al., 2016; Denais et al., 2016; Raab et al., 2016). It is well-established that Aβ42 can introduce membrane lesions and different non-excluding mechanisms were proposed, including membrane lipid interaction, alterations in membrane fluidity, pore formation, or lipid oxidation (Eckert et al., 2000; Muller et al., 2001; Ambroggio et al., 2005; Rauk, 2008; Axelsen et al., 2011; Viola and Klein, 2015). We speculated that αAβ42 would also trigger membrane lesions in our yeast system. To test this, we analyzed the plasma membrane integrity of wild-type and bro1Δ cells by cryo-EM. We chose the bro1Δ strain because here αAβ42wt and αAβ42G37C was almost lethal (Figure 5A) and because Bro1 is the yeast ortholog of human Alix, a proposed biomarker for AD (Sun et al., 2015). Consistent with our hypothesis, the cryo-EM study showed that the plasma membrane of bro1Δ cells expressing αAβ42wt seemed heavily corrugated while the bro1Δ cells transformed with empty vector displayed a more modest phenotype. In the wild-type strain, the plasma membrane remained ostensibly smooth even upon expression of αAβ42wt (Figure 5B). However, although we did not quantify, the observed membrane corrugating effects of specifically the bro1Δ strain expressing αAβ42wt are strikingly obvious. Moreover, when compared to the wild-type cells, the ER morphology in the bro1Δ mutant was completely different and appeared to be deteriorated. Indeed, only a minimal perinuclear and cortical ER was detected and the cells displayed the αAβ42-linker-GFP fusion mostly in filamentous structures and foci (Figures 5C,D). Thus, expression of αAβ42wt seems to dramatically affect all membranous structures evidencing that Bro1, and by extension the ESCRT system, is absolutely required for the repair of membrane lesions induced by αAβ42. In line with this essential requirement of ESCRT, we observed that while still seeing a tendency of increased PI uptake after 4 h induction for those bro1Δ cells expressing toxic αAβ42, a maximal PI uptake in all bro1Δ transformants is seen after 24 h, even for those strains expressing the non-toxic αAβ42L34T mutant or the ev-αGFP control (Figure 5E).
FIGURE 5. The role of Bro1 for membrane lesion repair. (A) Growth profile on galactose-containing medium of a strain deleted for the ESCRT-III accessory factor BRO1 transformed with an empty vector (ev-αGFP) or constructs allowing for expression of αAβ42wt or αAβ42G37C. Cryo-EM pictures (B) and fluorescence microscopy pictures (C) of wild-type and bro1Δ cells transformed with an empty vector (ev) or expressing αAβ42wt and grown for 6 h on galactose-containing medium. The indents in panel (B) zoom in on the plasma membrane and cell wall. The black arrowhead in (B) indicates a lipid droplet. Scale bars for cryo-EM pictures represent 200 nm. (D) BY4742 wild-type and a bro1Δ strains transformed with a plasmids carrying αAβ42wt and additionally a plasmid allowing the expression of Kar2(1-135)-mCherry-HDEL (ERCherry), a marker for the ER. DNA was stained with Hoechst. Cells were grown in medium allowing for gene expression for 6 h prior to microscopy. Scale bars for fluorescence pictures represent 2 μm. (E) PI staining of cells deleted for BRO1 transformed with constructs allowing for expression of αAβ42wt, αAβ42L34T, αAβ42G37C, or αGFP after 4 or 24 h growth on galactose-containing medium. Error bars represent standard deviations of at least four independent transformants.
The cryo-EM study also pointed to the formation of cortical vesicle-like structures, which we believe may correspond to lipid droplets. Since previous studies have shown that the formation of lipid droplets denotes an adaptive response to a chronic lipid imbalance (Vevea et al., 2015; Garcia et al., 2018), which is likely to occur in bro1Δ strain because of hampered MVB formation and lipid turnover, we decided to perform a Nile Red staining to visualize the droplets. While during the first hours of induction on galactose-containing medium we observed an overall enhanced lipid droplet biogenesis in all bro1Δ strains as compared to the respective wild-type strains, the increase was persistent and especially more dense droplets were seen with bro1Δ cells expressing αAβ42G37C or αAβ42wt (Figure 6A). This observation confirms previously reported data on an increased lipid droplet load in an Aβ42 wild-type strain (Chen et al., 2017).
FIGURE 6. αAβ42 instigated lipid droplet biogenesis. (A) Nile Red stainings (top panel), a marker for lipid droplets, of wild-type and the bro1Δ cells expressing αAβ42wt, αAβ42G37C, or αAβ42L34T or αGFP after 2, 4, or 6 h growth on galactose-containing medium. The bottom panel (yellow) shows magnifications and deconvolved parts of the top-panel pictures at time point of 6 h. Note that due to deconvolution the picture intensities are enhanced. Scale bars represent 2 μm. (B) DHE staining of cells deleted for BRO1 transformed with constructs allowing for expression of αAβ42wt, αAβ42L34T, αAβ42G37C, or αGFP after 4 or 24 h growth on galactose-containing medium. Error bars represent standard deviations of at least four independent transformants.
The perturbation of lipid homeostasis has also been linked to defects in the ER and mitochondria (Vevea et al., 2015), and the latter incited us to monitor the level of ROS in the bro1Δ strains. As shown, and comparable to the PI uptake, we found similar high levels of ROS in all strains tested indicative that this is a characteristic mainly associated to the deletion of BRO1 itself (Figure 6B).
During the past decades, several studies validated the use of yeast to decipher the pathobiology underlying a variety of human disorders. Especially for degenerative protein folding diseases, like Huntington's, Parkinson's, or Alzheimer's disease, this led to the discovery of processes and molecular pathways contributing to cell demise (Winderickx et al., 2008; Franssens et al., 2010; Swinnen et al., 2011; Porzoor and Macreadie, 2013; Tenreiro et al., 2013, 2017; Chen and Petranovic, 2016; Jiang et al., 2016; Verduyckt et al., 2016; Fruhmann et al., 2017). The insight gained from these studies were not only relevant in the context of disease, but they also clarified fundamental aspects on how a cell manages to maintain proteostasis and the consequences in case this system fails. In this paper, we used a previously reported model to study the repercussions when the APP-derived peptide Aβ42 is expressed in yeast (D'Angelo et al., 2013; Vignaud et al., 2013). Though this model makes use of a GFP fusion, the use of the super-toxic Aβ42G37C and the non-toxic Aβ42L34T mutants clearly demonstrated that the properties of the GFP fusion are dictated by the Aβ42 peptide moiety. Furthermore, by comparing constructs with or without an N-terminal fusion with the α-prepro sequence and conditions that retain the α-prepro in the ER, it became obvious that the processing in the ER/Golgi system and the subsequent shuttling into the secretory pathway is essential to unleash the full toxic capacity of the Aβ42-linker-GFP fusion. Our data also contradict the argument that the αAβ42 instigated toxicity would simply be due to an overload of the ER/Golgi processing system because both wild-type and mutant αAβ42 are processed in the same manner (D'Angelo et al., 2013; Vignaud et al., 2013) and no toxicity is seen in case of expression of mutant αAβ42L34T.
The use of our yeast model allowed us to confirm some data previously reported. This included the impact of αAβ42 on endocytosis (Treusch et al., 2011; D'Angelo et al., 2013), where we now show that strains with a deletion of SAC6 or RVS167 display an increased αAβ42 toxicity, as well as the impact on mitochondrial functioning (D'Angelo et al., 2013; Chen and Petranovic, 2015; Chen et al., 2017), which we illustrated by co-localization studies and the observation that αAβ42 enhances ROS formation. These data are relevant because, indeed, changes in endocytic capacity and mitochondrial dysfunction are typically seen in the pathogenesis of AD and have been observed in other AD models as well (Thomas et al., 2011; Wang et al., 2011; Avetisyan et al., 2016; Schreij et al., 2016; Dixit et al., 2017; Shoshan-Barmatz et al., 2018; Xu et al., 2018). However, the observed partial co-localization of toxic αAβ42 with mitochondria may either indicate a direct interaction of the peptide with this organelle or it may simply be a reflection of the interaction between the ER and mitochondria through the membrane contact sites known as ERMES (Kornmann et al., 2009). This aspect needs to be analyzed in more detail. Interestingly, mitochondria associated membranes (MAMs), the mammalian counterpart of ERMES, have already been implicated in AD (Area-Gomez and Schon, 2016; Paillusson et al., 2016). Closely related to the observed mitochondrial dysfunction is our observation that deletion of AIF1, encoding a cell death effector, has a protective effect on the αAβ42 expressing yeast cells. This suggests that αAβ42 may induce an apoptotic-like program in yeast, which fits the finding that neuronal cells die through apoptosis in AD (Ohyagi et al., 2005; Shoshan-Barmatz et al., 2018). Our data also show that particularly the expression of the toxic αAβ42 isoforms is linked to the formation of ER-associated foci and filamentous structures. Though we did not study these structures in detail and previously believed these to represent vesicles (D'Angelo et al., 2013), it is well-possible that they may in fact be ER aggregates or clustering of ER membranes, which are both indicative for ER stress (Varadarajan et al., 2012, 2013; Vevea et al., 2015). Also ER stress is associated to AD (Gerakis and Hetz, 2018) and several links between ER stress and mitochondrial dysfunction have been proposed in this neurodegenerative disorder (Costa et al., 2012; Barbero-Camps et al., 2014; Erpapazoglou et al., 2017).
One of the most striking observations made in our studies is the role of ESCRT in modulating the αAβ42 toxicity. The ESCRT system functions in the MVB pathway and several studies have linked this role of ESCRT to AD. Neurons of AD transgenic mice were shown to display enlarged MVBs as compared to the neurons of wild-type mice, and ESCRT was demonstrated to modulate intracellular Aβ42 accumulation by directing APP to lysosomal degradation and by enhancing Aβ42 secretion. In addition, ESCRT components were found associated with amyloid plaques in transgenic mice and to granular structures hippocampal neurons of AD diseased human brain (Yamazaki et al., 2010; Edgar et al., 2015; Willen et al., 2017). However, apart from its function in the MVB pathway, ESCRT is also required for the repair of membrane lesions. Here, both cryo-EM and PI-staining give a strong impression of the presence of such lesions at the plasma membrane of cells lacking the ESCRT component Bro1, probably explaining in part the sick phenotype of the bro1Δ mutant. As such, our data strongly suggest that the role of ESCRT for plasma membrane repair, which so far was only demonstrated in mammalian cells, is evolutionary well-conserved. Interestingly, we found that the disruption of the plasma membrane integrity in the bro1Δ strain is dramatically exacerbated upon expression of αAβ42 and that this came along with the deterioration of the ER and an almost lethal phenotype. This is intriguing for several reasons. It demonstrates that when the fully processed Aβ42-linker-GFP arrives at the plasma membrane, the peptide further aggravates plasma membrane disruption, which given the observed effect on the ER, might well be involving fusion of secretory vesicles that contain disordered membranes. The fact that our screens retrieved the KO strain lacking SSO2 as suppressor of αAβ42 toxicity favors the last possibility. Indeed, SSO2 encodes a plasma membrane t-SNARE that is required for fusion secretory vesicles (Grote et al., 2000). Moreover, the data recapitulate observations made for AD where, as mentioned, Aβ42 was shown to introduce membrane lesions via different non-excluding mechanisms, including membrane lipid interaction, alterations in membrane fluidity, pore formation, or lipid oxidation (Eckert et al., 2000; Muller et al., 2001; Ambroggio et al., 2005; Rauk, 2008; Axelsen et al., 2011; Viola and Klein, 2015).
Given the effect of Aβ42 on plasma membrane integrity, the presence of this peptide also impacts on the overall cellular lipid homeostasis. In fact, extensive lipid alterations are implicated in the AD disease pathology but it is still a matter of debate whether such alterations are the cause or the consequence of AD (Grosgen et al., 2010; Xiang et al., 2015; El Gaamouch et al., 2016). In yeast, the expression of Aβ42 has been linked to a transcriptional upshift of key regulators of lipid metabolism as well as an enhanced formation of lipid droplets (Chen et al., 2017). Our screens with the yeast deletion collection and our Nile Red stainings support the link between Aβ42 and lipid metabolism. Intriguingly, a recent study demonstrated lipid droplet formation to be an adaptive response to an acute lipid imbalance in yeast cells. The same study then also showed that the biogenesis of these droplets occurs at ER aggregates (Vevea et al., 2015), which we believe to correspond to the ER-associated foci and filamentous structures seen when yeast cells express toxic forms of αAβ42, as mentioned. Notably, also in transgenic mouse models of AD an enhanced lipid droplet formation is observed (Hamilton et al., 2010; Yang et al., 2014), again underscoring the relevance of the data obtained in yeast.
GF, CM, HV, MV, and NT performed experiments and analyzed data. GF and JW wrote the draft. CDV, CC, and JW contributed conception and design of the studies. CC and JW corrected and edited the draft.
This work was financially supported by the KU Leuven and the FWO Vlaanderen (Project No. G0A6315N). CDV was supported by the Swiss National Science Foundation and the Canton of Fribourg.
We want to thank Joëlle Rosseels, Dorien Vliegen, and Elodie Cougouille for technical and experimental support. We also want to thank Dr. B. Salin and to the Bordeaux Imaging Center (BIC) for performing TEM experiments.
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2018.00406/full#supplementary-material
^https://www.yeastgenome.org/cgi-bin/GO/goSlimMapper.pl
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Keywords: Aβ42, amyloid beta, Alzheimer's disease, ESCRT, membrane repair, Saccharomyces cerevisiae, yeast
Citation: Fruhmann G, Marchal C, Vignaud H, Verduyckt M, Talarek N, De Virgilio C, Winderickx J and Cullin C (2018) The Impact of ESCRT on Aβ1-42 Induced Membrane Lesions in a Yeast Model for Alzheimer's Disease. Front. Mol. Neurosci. 11:406. doi: 10.3389/fnmol.2018.00406
Received: 13 April 2018; Accepted: 16 October 2018;
Published: 05 November 2018.
Ralf J. Braun, University of Bayreuth, Germany
Md. Golam Sharoar, University of Connecticut Health Center, United States
Markus Babst, The University of Utah, United States
Copyright © 2018 Fruhmann, Marchal, Vignaud, Verduyckt, Talarek, De Virgilio, Winderickx and Cullin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Joris Winderickx, joris.winderickx@kuleuven.be Christophe Cullin, christophe.cullin@u-bordeaux.fr
†These authors have contributed equally to this work
‡Co-first authorship


Oral hygiene: all your questions answered!
Chocolate: how good and bad is the world's favorite treat?
A guide to staying fit and healthy in middle age - When you get older, you become more aware of the toll that poor health decisions take on your body. A sedentary lifestyle can decrease life expectancy and well-being, which is why it is important to stay active and mindful of diet and fitness choices.
Middle age health - After 50, it's easy to get relaxed when it comes to taking care of your body, but a lack of fitness and an unbalanced diet can pose a great risk for your overall health, as reported by the BBC.
Balanced diet - Eating well should be at the center of your health concerns. A balanced diet that is rich in nutrients and vitamins will boost your energy and strength levels, as well as your immune system.
Supplements - Avoid supplements and rely mainly on nutrients you're getting from your diet choices. Supplements are not only expensive, but they can also have a negative effect on your health, according to the US National Institutes of Health.
Probiotics - Probiotics are great as they reduce inflammation caused by microbial activity in your gut, which in turn improves skin-friendly flora that fights ageing symptoms.
Coffee and tea - Too much caffeine can cause dehydration, but moderate coffee consumption can boost physical performance and is thought to reduce the risk of Alzheimer's and dementia, as shown in a study by the University of Eastern Finland.
Go out - As great as it is to go to the gym or attend other indoor lessons, taking nature walks can bring mental health benefits, and is a great way to clear your mind and get moving at the same time.
How much physical activity do you need? - Adults can substantially improve their health with 2.5 hours of moderate cardio a week, or 75 minutes of vigorous activity a week, according to Healthy Midlife.
A busy life is no excuse - That is equivalent to 10 to 20 minutes of exercise a day, depending on the intensity, so there's no excuse anymore!
Start slowly - It's important to start slowly to avoid injuries, so it might be a good idea to get help from a certified personal trainer.
Muscle strengthening - Our muscle strength and mass decline with age, which is why muscle training is key when exercising in middle age. It prevents osteoporosis and helps keep your metabolism at a high rate.
Moderate aerobic activities - A moderate aerobic activity should be lightly challenging—you should breathe fast without being out of breath, and you should start sweating a bit after 10 minutes. This is the ones beginners should go for.
Vigorous aerobic activities - A vigorous aerobic activity should be physically challenging and demanding—you should be breathing rapidly and intensely, as with jogging or running. These are adequate for fitter individuals.
Find something you really enjoy - Finding a sport you really like practicing is halfway to fitness happiness. That way, you don't feel like you're being forced to workout—you're just having fun!
Swimming - Swimming is a great low-impact exercise that stimulates all the big muscle groups while being gentle on your joints.
Find a fitness partner - Having a workout partner might give you the motivation you need to get started, as you can commit to goals together and stick to them.
Be an early bird - Your metabolism tends to slow down with age, but if you opt for exercising in the morning, it will stay boosted for longer.
Set a goal - Setting specific long-term or even short-term goals is key during your fitness journey. Make them attainable and measurable so you don't get demotivated in the process.
Buy your own kit - The gym isn't the only option. In fact, you can save a lot of money by exercising at home or outdoors. A fitness tracker and good sports gear is all you need to become autonomous.
Flexibility - Flexibility training is essentially for middle-aged people as your joints and muscles become stiffer with age. Yoga is great for enhancing your flexibility, so give it a try!
Mental fitness - Yoga is not only good for your body, but also for your mind. Mental fitness and stability is as important as physical exercise, so include relaxing workouts as well as meditation in your weekly routine to achieve better overall health.
Restorative sleep - Try to go to bed at the same time every night so your body can develop natural habits and rest properly. Quality sleep has an effect on both physical and mental health.
Eating before bedtime - Eating a heavy meal before going to bed affects the quality of your sleep, as the body struggles to digest and sleep at the same time.
No cigarettes or alcohol - It's no news that cigarettes and alcohol are bad for your health, but they're even more damaging at an older age. According to The Telegraph, studies show that people who quit smoking before the age of 44 can live almost as long as those who never smoked.
Aspirin - A low dose of daily aspirin has been linked to a lower risk of heart attack and stroke, but it can increase the risk of developing stomach ulcers, so check with your doctor before you self-medicate.
Skincare - Ageing skin is one of the most evident symptoms that comes with growing older, but it can be delayed as much as possible by adopting the correct skincare routine. Check with your dermatologist which products work best for you.
Posture - Poor habits throughout your life can lead to terrible posture at an older age. Try to sit and stand straight as much as possible, and practice exercises like Pilates and yoga to improve your posture.
Pets - Getting a pet is a great way to get you out of a sedentary lifestyle. A pet not only provides company, but it is also known for having positive impacts on your lifestyle, both physically and mentally.
Walk everywhere - The first and most crucial step to getting fit is movement. Walking increases stamina and reduces stress levels, so try to do it as much as you can.
Most people look forward to a relaxing lifestyle as they get older, but that doesn't have to mean being sedentary. In fact, when crossing the middle age threshold, we ought to be more careful with our life choices in order to maximize our health and overall well-being.
Are you in need of a midlife health check? Click through this gallery for the best tips on how to stay fit and healthy.


text:
A Badlands Baptism
(idea) by dem bones Thu Mar 30 2000 at 0:22:58
We're outside of the town now, heading to a place in the Badlands that I remember. It's supposed to be part of a national preserve, but I know of an access road that will cut us into there. Inside is a clear river-stream that I want to bring us all into. Passing through, with our vehicles across the way; as dumb as it sounds, I think we need to be purified. And this place just seems right. And who am I to say what is dumb and what is not? This a pilgrimage, literally and figuratively. Perhaps this will be what is needed.
The landscape looks like the Negev, or Mare Imbrium. Driving along a barely passable two track road, I skirt ahead on the cycle, feeling it throb like some sort of overgrown vibrator between my legs. My ass hurts from the vibration. The road is slightly grown over, but I see the bridge ahead, and no rangers around. I drive across the I-beam bridge, and wait idling. Ryan, Jeff and Scott get out, and Rob and Chris get out of the respective vehicles, looking furtively about. Rob still looks worried about the letter. Jhasen had sought to explain in terms of Steve, but I think it has gone to a more personal field. It's bringing him to stop Steve, but also bringing us to a level we didn't know about. Jesus, it sounds like Olympic training. Nate and Sally come out, with Cliff, after checking the body. When we go to Wall Drug, I remind myself to get a side-car. It's lonely out there, with disembodied voices echoing in my helmet. Besides, the bus has begun to stink like an Australian table wine. He could use a good airing. Walking across the bridge, small talk is made, and the mood is not reverent, or expectant, but almost like everyone knows this is what they have to do. This isn't a question any longer.
We go at our own pace. I have found a smooth rock, and sit, letting the spring water feed over me. Sally has gone through, and sits on the bank, not really saying anything, but looking wistfully at the sky. Chris and Rob have begun to play in the stream, like some sort of monstrous children's playground experiment gone mad. Ryan, Cliff and Scott are standing, letting the water curl around their calves, watching the behemoths at play. Cliff seems to be more detached-perhaps he's thinking about Mary Beth. Or killing Purple Dave. Either way is good for the soul. Close to him, Ryan and Scott are talking quietly about Wall Drug. They seem to be even more inseparable then normal. Nate has taken a sitting position on the bridge, reading Bradbury and not really joining the games. Understandable. Jeff just looks confused by all this, like something has changed from Northern Michigan. Lots of people he doesn't know. Empathy.
As we all come out, it feels good to be clean again. Physically good. I hop into the bus, and send this missive out, hoping Tracy, Nikki et al. Are reading this.
-hemos-
--Letters from a Savior; Offer for a few--
back--forward
Prairie Fires Letters from a Savior; Offer for a few Dead Hand Writing The Badlands
Windows left open not for the breeze, for moonlight October 17, 2001 evidence of moonlight The Letter
Steve Lawrence Olympic Missive pilgrimage
Total Depravity Ray Bradbury Negev Add Me On
Gilgamesh Great Wall Hemos Spring water
Purify Preserve Inseparable
bhikkhuni
They shared a love of impermanence, briefly

redpajama_set_name: RedPajamaCommonCrawl


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January 22, 2018 in new episode
This week: there's a new religious version of Wikileaks, Michael Wolff's Fire and Fury is flying off the shelves, South Korean universities have reached a deal with a science journal publisher, garbage collectors have started a library of discarded books, President Trump announced the Fake News Awards, Prisons face backlash over book censorship, and schools are teaching kids to spot fake news. All this, plus the New York Times Bestsellers, the Amazon Charts, and the latest news from aois21, Literally This Week.
Tags: Literally this week, podcast, aois21 audio, news, literature, books, reading, libraries, publishing, publishers, bookstores, Wikileaks, faithleaks, California, Azerbaijan, UCLA, Harry Potter, f-bomb, Sam Rockwell, Fire and Fury, Michael Wolff, CLPE, Jeff Bezos, Amazon, Stephen King, PEN Award, South Korea, elsevier, Handmaid's Tale, T S Eliot Prize, Apple News, Facebook, garbage, CreateSpace, Overdrive, public libraries, One Million Moms, Scholastic, Open Access, science journals, adaptaing, adaptation, fake news, Fake News Awards, President Trump, voice, bestsellers, New York Times, Amazon Charts, New York Times Bestsellers
May 30, 2016 in new episode
This week: Judy Blume has opened her own bookstore, Simon Cowell is writing a children's book, big publishers are on the decline, the daughter of a Hong Kong bookseller wants the U.S. to help, realtors are waiting on Amazon, the National Book Festival poster was released, and the European Union is making all publicly funded scientific articles free. All this, plus the New York Times Bestsellers, Literally This Week. This episode is sponsored by Interlude to Sentimental Me! the new poetry collection from Michael B. Judkins now available for preorder from aois21 publishing.
Tags: Judy Blume, bookstores, indies, indie booksellers, marketing, Becky Fincham, JK Rowling, Chinese poetry, translation, sports day, sports books, Australia, National Archives, Cuba, restoration, scientists, simon cowel, children's book, Gabriel Garcia Marquez, ashes, Colombia, Mexico, Ta-Nehisi Coates, Brooklyn, Ed Riche, political correctness, Library of Congress, subject headings, U.S. Congress, objection, Zero K, Don DeLillo, Big publishing, publisher, Jungle book, competition, Hay Festival, Marlon James, Rosario Castellanos, poets, Boston Public Library, Jill Bourne, Stephen King, Cheryl Strayed, Donald Trump, Simon Armitage, list-making, poetry, Hong Kong, bookseller, Amazon, ComiXology, Subscription, Enid Blyton, Famous Five, geo-blocking, eBooks, Macmillan, Pronoun, Vook, Flatiron Building, CRE, TS Eliot, George Orwell, Animal Farm, Chinese millennials, Karl Marz, Karl Marx, Hip hop, Apple, Bank Holiday, New author giveaway, Pop culture, coloring books, French strike, work email, National Book Festival, poster, Journey to the Unknown, Dickens, Virginia Woolf, Charles Dickens, Nathaniel Philbrick, interview, Susan Rukeyser, redemption, Kris Bertin, bartending, Alaska, Kenai Community Library, vanadalism, Mars, Scientific Journals, European Union, Overdrive, summer reading program, Dorothy and Lewis B. Cullman Center for Scholars and Writers, New York Public Library, Audiobooks, Self-published, self-published ebooks


text:
Lucy A. Snyder
Author • Editor • Writing Instructor
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What is Gaslighting?
March 2, 2017 Lucy A. Snyder writing 0
I've worked with a number of horror writers, and many of my students' manuscripts deal with characters who are in emotionally or psychologically abusive situations. It's critical to portray these situations realistically, and sometimes my students don't fully understand key concepts related to psychological abuse; gaslighting is one of them.
Gas Light was a 1938 stage play that was adapted into movies in 1940 and 1944; the plot centers on a man who tries to drive his wife insane by changing the environment of their house in small but perceptible ways while insisting to her that everything is the same. Namely, he dims the gas lights of the house; she notices it, but he insists it's all her imagination.
Gaslighting is a form of psychological and emotional abuse. It's a typical tactic used by sociopaths and people with narcissistic personality disorder. Most times, it doesn't actually involve the physical environment manipulations portrayed in the play and movies.
Typically, Person A uses various types of emotional manipulation to convince Person B that his or her authentic emotional reactions to an injustice or injury caused or abetted by Person A are crazy or unreasonable, or that the injury never even happened. The result is that Person A doesn't have to change (or even feel remorseful about) his or her behavior, because a successful gaslighting causes Person B to doubt and second-guess his or her own emotions, perceptions and judgements. Ultimately, Person B will go silent about whatever bad thing that Person A keeps doing.
There are also instances in which one person gaslights another for the sadistic amusement of seeing another person distressed and confused. These situations often do involve physical environment manipulations, which the instigator will trivialize as harmless pranks if he or she is discovered staging them. Imagine, for instance, the wife of an elderly man with mild dementia who repeatedly hides his beloved pocket watch and then tells him he lost it, sending him on frantic, fruitless hunts through their house while his wife quietly plants the watch someplace he already searched thoroughly.
Children suffering from sexual abuse are frequently subjected to gaslighting by their abusers, as are rape victims and domestic violence victims.
Here's a typical gaslighting exchange:
Amy: "I wish you would stop flirting with other women at parties."
Joe: "What? What are you even talking about?"
Amy: "Bella. You were flirting with her."
Joe: "Christ, Amy, no I wasn't. God. You are so jealous; it's just ridiculous."
Amy: "I saw you kiss her!"
Joe: "No you didn't. All I did was lean in to tell her something about our boss that I didn't want Judy hearing."
Amy: "Judy was on the other side of the room –"
Joe: "No she wasn't. She was right there. You should get your eyes checked. Or maybe it was all that tequila you put away, huh? Christ, you are so unreasonable."
Amy: "But –"
Joe: "I'm so sick of your jealousy. Can't you just relax and let me enjoy a party without trying to control every move I make? Jesus. Why do you have to try to ruin everything?"
Amy: "I'm sorry. I just … nevermind."
Amy starts off very reasonably expressing her desire that Joe stop doing something hurtful to her. Joe comes back at her with a "the best defense is a good offense" style gaslighting, and he is certainly offensive. If Joe were merely trying to cover his ass about the incident, he might go with a standard apologetic lie: "I'm sorry, dear, I didn't know what I was doing and I'd never intentionally embarrass you like that, honest!" But here he completely denies what he did and abusively pretends it's all a figment of her crazy, jealous mind. He paints himself as the injured party and browbeats Amy into apologizing to him. Joe doesn't care if Amy really believes him or not; he's satisfied thinking he's punished her so much for bringing up his infidelity that she won't protest the next time he does something.
Sarah: "I … I need to talk to you about Bob, that new guy in your gaming group."
Mike: "Sure, what's up?"
Sarah: "We went to college together, and once we were at a party, and …."
Mike: "And what?"
Sarah: "And I had too much to drink, and I went to lie down in a back room, and when I woke up Bob was on top of me, and he'd gotten my jeans off –"
Mike: "No way! Bob's such a nice guy; he'd never do something like that."
Sarah: "Yes, I know he seems nice, but he did it, and … I'm not comfortable with him in the house."
Mike: "That's crazy. Why would Bob need to pull that kind of creeper stuff? He's good-looking, he's got a kick-ass job, and the jokes that guy tells are hilarious."
Sarah: "I know, but –"
Mike: "Come on. You were drunk, right? It probably wasn't even Bob that did it. And besides, that was what, five years ago? Aren't you over it yet?"
Sarah: "I …."
Mike: "Aw, why are you crying? There's nothing to cry about!"
Sarah: "He tried to rape me. He would have done it if Mark hadn't come in and interrupted him."
Mike: "You're so emotional. Come on. Bob's not some scary rapist; he's a good guy. He's the best GM we've ever had! I'm sure that even if all that happened, he's totally changed."
Sarah: "But –"
Mike: "But nothing. He's fine. You're fine. You just need to get over this weird emotional thing you've been harboring against him all these years."
Sarah: "He –"
Mike: "Enough, already! He's coming to the house with the guys next Tuesday. Just make us some sandwiches and be cool, okay?"
Sarah: "…"
Mike: "Come on, Sarah. You better not cause a bunch of drama and embarrass me in front of my friends."
Sarah: "Okay."
Mike never wants Sarah to suffer emotionally; the problem is that he just doesn't really care if she suffers as long as he gets what he wants. Defending her right to feel safe in her own home would involve disrupting his own social activities and saying "No" to his buddies, and Mike's too selfish and cowardly to do the right thing. Mike's also emotionally invested in the idea of Bob as a Really Cool Guy, because otherwise he'd have to admit he's been thoroughly tricked by a predator. From his perspective, it's easier to pretend that his wife is overly emotional and irrational than to address the problem that he's friends with a rapist. This exchange, unfortunately, is a pretty classic example of how rapists enlist other men in their social circles to help keep their victims quiet.
Who's Mary Sue?
A Writer's Guide to Evaluating and Attending Conventions
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How to comfort someone whose mother or father has died
Book Review: The Time Machine by H.G. Wells
Book Review: Lord of the Flies by William Golding
Roll of Thunder, Hear My Cry
Playing Poker With Tarot Cards: Assumption
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Recently constructed a new drive way to a mountain view spot on our acreage in Grizzly Valley. The future plan is to build a guest home for visiting family and friends (we also live in a small home ~400 sq f that is CROWDED when visitors come), but with so many other projects on the go we are postponing development.
Outhouse provided. Power can be supplied in the winter to plug vehicles in and wifi could be shared if interested – would add some extra cost.
There is a water filling station for blue jugs and large tanks at Deep Creek – 7 minute drive. Showers at Takhini Gas Station are lovely, and cheap!
Our place is located 45km from downtown Whitehorse near Lake Laberge.
We both work full time and would be interested in carpooling if schedules are similar.
Asking $300/month (wifi and power to be negotiated) but open to offers.
Pm or message here if you are interested.


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Home Showbiz Music 201001
T-Pain and Elton John On Grammy Awards Line-Up
/ Music, Showbiz
A bunch of acclaimed musicians have been added to the already star-studded line-up of the 52nd Annual Grammy Awards. Katy Perry, T-Pain and Elton John are the latest names thrown to the stellar ensemble.
While Katy is set to present award, T-Pain and Elton will be singing. Other celebrities booked to announce winners of the awards show are Alice Cooper, Placido Domingo, Wyclef Jean, Juanes, Miranda Lambert, John Legend, Lionel Richie and Seal. Movie director Quentin Tarantino and "Glee" actress Lea Michele are also among the latest presenters.
As for the new musical acts, there are Slash, Doug E. Fresh, Roberta Flack and Jennifer Nettles of Sugarland. Also, Jeff Beck and Imelda May will make a duet for a tribute show for guitar virtuoso Les Paul, and Green Day will take the stage with the cast of "American Idiot" play.
This year, Grammys will be aired live from Los Angeles' Staples Center on January 31. Jonas Brothers, Ke$ha, Justin Bieber, Eminem, Lil Wayne, Drake, Miley Cyrus, Kristen Bell, Josh Duhamel, Mary J. Blige, Lady GaGa, Taylor Swift and Beyonce Knowles are previously-announced performers and presenters.
Source: aceshowbiz
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Breweries will bring their beer to several tasting stations along the trails, pouring samples as skiers and snow showers pass through.
The event has grown to include the following North Country and Adirondack breweries: Big Tupper Brewing, Raquette River Brewing, Valcour Brewing Company, Township 7 Brewing Company, Oval Brewing, Lake Placid Pub & Brewery, Big Slide Brewery and Public House and High Peaks Distribution, which is bringing Livingood's Restaurant and Brewery, and Saranac Beer. MAC's safe ride will be operating from noon to 3 pm to provide transportation to participants.
The event is Saturday, February 24 from 1 to 4 pm at 141 Country Club Road, Tupper Lake. The beer trail pass is $10 per person. Photo ID is required.
Also happening simultaneously is the Fire & Ice Golf Tournament from noon to 4 pm on the golf course. Three person teams will play the five-hole course twice. The cost is $60 per team with the proceeds to benefit the Lion's Club community projects.
Photo of Tupper Lake Brew Ski provided.


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Que. Statement: In Bombay, railway trains are indispensable for people in the suburbs to reach their places of work on time.
I. Railway trains are the only mode of transport available in the suburbs of Bombay.
II. Only railway trains run punctually.
a. Only assumption I is implicit
b. Only assumption II is implicit
c. Either I or II is implicit
d. Neither I nor II is implicit
Correct Answer:Only assumption II is implicit
Statement: In spite of less than normal rainfall in the catchment areas during the first two months of monsoon of the lakes supplying water to the city the authority has not yet affected any cut in the water supply to the city.
I. The rainfall during the remaining part of the monsoon may be adequate for normal water supply.
II. The present water level of the lakes supplying water to the city may be adequate for normal supply.
Statement: Of all the radio sets manufactured in India, the 'X' brand has the largest sale.
I. The sale of all the radio sets manufactured in India is known.
II. The manufacturing of no other radio set in India is as large as 'X' brand radio.
Statement: If you have any problems, bring them to me.
I. You have some problems.
II. I can solve any problem.
Statement: Deepak has a large collection of books and he keeps on purchasing new books to add to his collection.
I. Deepak loves and cares for books.
II. Deepak has read each and every book that he has purchased.
Statement: "In order to bring punctuality in our office, we must provide conveyance allowance to our employees." - In charge of a company tells Personnel Manager.
I. Conveyance allowance will not help in bringing punctuality.
II. Discipline and reward should always go hand in hand.
Statement: The X passenger car manufacturing company announced a sharp reduction in the prices of its luxury cars.
I. There may be an increase in the sale of luxury cars of Company X.
II. The other such car manufacturers may also reduce their prices.
III. The competitor companies may not reduce their prices.
Statement: "If you are a mechanical engineer, we want you as our supervisor." - An advertisement by Company X.
I. Mechanical engineers are expected to be better performers by Company X.
II. The Company X needs supervisors.
III. Mechanical engineers may get attracted and apply to Company X.
Statement: 'Guests should be provided lunch.' - A tells B.
I. Unless told, lunch may not be provided.
II. Guests will stay during lunch time.
Statement: "As there is a great demand, every person seeking tickets of the programme will be given only five tickets."
I. The organizers are not keen on selling the tickets.
II. No one is interested in getting more than five tickets.
Statement: 'Double your money in five months.' - An advertisement.
I. The assurance is not genuine.
II. People want their money to grow.
Statement: The higher echelons of any organization are expected to be models of observational learning and should not be considered as merely sources of reward and punishments.
I. Employees are likely to be sensitive enough to learn by observing the behaviour of their bosses.
II. Normally bosses are considered as sources of reward and punishment.
Statement: "You should not grant him leave in this week due to exigency of work." - A supervisor advises the administrative officer.
I. Request for leave can be turned down also.
II. The supervisor has reviewed the work required to be done during the said period.
Statement: If it is easy to become an engineer, I don't want to be an engineer.
I. An individual aspires to be professional.
II. One desires to achieve a thing which is hard earned.
Statement: The Principal instructed all the teachers to be careful in class because some students may disturb other students.
I. The teachers will handle the situation properly and they will point out the naughty students.
II. The students will welcome the decision of the Principal.
Statement: Lock your valuables in a cupboard and call everybody gentleman.
I. Valuables locked in cupboard cannot be stolen.
II. Stealing is a crime.
Statement: Unable to manage with the present salary, Arun has decided to join another company.
I. The new company has better work environment.
II. The present company offers moderate pay packets.
III. The new company offers higher salary to all its employees.
Statement: "It has become a necessity to computerize all the functions of our Institute to maintain the present position." - A statement of the Director of XYZ Institute.
I. Unless computerized, the Institute will fall behind the race.
II. The functions of the Institute are too complex to be handled manually.
Statement: The university Authority has decided to decentralize conduct of terminal examinations and give this responsibility to each college for its students to avoid delay in declaration of results.
I. The colleges are equipped to carry out this responsibility.
II. There may not be uniformity in evaluation standard across the colleges.
III. The students may welcome this new development.
Statement: "According to me, you should get your child examined by a specialist doctor." - A tells B.
I. Specialist doctors are able to diagnose better than ordinary doctors.
II. B will certainly not agree with A's advice.