FEMS Yeast Research, 15, 2015, fov061
doi: 10.1093/femsyr/fov061
Advance Access Publication Date: 7 July 2015
Research Article
RESEARCH ARTICLE
Amyloid-β peptide-induced cytotoxicity and
mitochondrial dysfunction in yeast
Xin Chen1 and Dina Petranovic1,2,∗
1
Systems and Synthetic Biology, Department of Biology and Biological Engineering, Chalmers University of
Technology, SE-412 96 Göteborg, Sweden and 2 Novo Nordisk Foundation Center for Biosustainability,
Chalmers University of Technology, SE-412 96 Göteborg, Sweden
∗ Corresponding author: Novo Nordisk Foundation Center for Biosustainability, Chalmers University of Technology, SE-412 96 Göteborg, Sweden.
Tel: +46-31772-3836; Fax: +46-31772-3801. E-mail: [email protected]
One sentence summary: The authors have established a yeast Aβ model in which constitutively producing native Aβ exhibited a lower growth yield,
lower biomass yield, increased oxidative stress and hallmarks of mitochondrial dysfunction.
Editor: Cristina Mazzoni
ABSTRACT
Alzheimer’s disease (AD) is the most common neurodegenerative disease, characterized by deposits of amyloid-β (Aβ)
peptides. However, the underlying molecular mechanisms of neuron cell dysfunction and cell death in AD still remain
poorly understood. Yeast Saccharomyces cerevisiae shares many conserved biological processes with all eukaryotic cells,
including human neurons. Thanks to relatively simple and quick genetic and environmental manipulations, the large
knowledge base and data collections, this organism has become a valuable tool to unravel fundamental intracellular
mechanisms underlying neurodegeneration. In this study, we have used yeast as a model system to study the effects of
intracellular Aβ peptides and we found that cells constitutively producing native Aβ directed to the secretory pathway
exhibited a lower growth rate, lower biomass yield, lower respiratory rate, increased oxidative stress, hallmarks of
mitochondrial dysfunction and ubiquitin–proteasome system dysfunction. These findings are relevant for better
understanding the role of Aβ in cell stress and cell damage.
Keywords: Amyloid-β; Alzheimer’s disease; yeast; mitochondria; oxidative stress; ubiquitin-proteasome system
INTRODUCTION
Alzheimer’s disease (AD) is the most common progressive neurodegenerative disease and is responsible for 60–80% of dementia cases (Ferri et al. 2005). It affects approximately 36 million
people worldwide, and due to increasing longevity of the human population will become the world’s leading cause of death
by 2050 (Williams 2009). AD is pathologically characterized by
the presence of extracellular plaques comprised of amyloid-β
(Aβ) peptide (Hardy and Selkoe 2002). Aβ peptides (ranging in
length from 38 to 43 amino acids) are proteolytic products of the
amyloid precursor protein (APP) and are sequentially cleaved by
β- and γ-secretases (Selkoe 2001). Mutations in the APP protein
or the γ-secretases cause familial AD, and the mutations direct
the APP cleavage towards the Aβ42 peptide production, that predominates in cerebral plaques. Aβ42 peptide is more hydrophobic and thus more prone to fibril formation (Selkoe and Wolfe
2007) and increasing evidence suggests that small oligomers of
Aβ are the most toxic species (Hartley et al. 1999; McLean et al.
1999).
In neurons, the cleavage of APP to generate the Aβ peptide occurs within the secretory and endosomal pathways (Thinakaran and Koo 2008). Although the extracellular amyloid
plaques and oligomeric species of Aβ are considered important
hallmarks of AD, emerging evidence from transgenic mice experiments and studies of patients in the clinic indicates that the
Received: 23 March 2015; Accepted: 1 July 2015
C FEMS 2015. All rights reserved. For permissions, please e-mail: [email protected]
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FEMS Yeast Research, 2015, Vol. 15, No. 6
Table 1. Plasmids and strains used in this study.
Plasmid or strain
Plasmid
p416 ADH
p416 TEF
p416 GPD
p426 ADH
p426 TEF
p416 ADH-Kar2-Aβ42
p416 TEF-Kar2-Aβ42
p426 ADH-Kar2-Aβ42
p416 GPD-Kar2-Aβ42
p426 TEF-Kar2-Aβ42
p416 GPD-Kar2-Aβ40
Strain
CEN.PK 113–11C
P416 EP
P426 EP
p416 ADH-Aβ42
p416 TEF-Aβ42
p426 ADH-Aβ42
p416 GPD-Aβ42
p426 TEF-Aβ42
p416 GPD-Aβ40
Relevant genotype
Reference
CEN, ADH1 promoter, URA3 marker
CEN, TEF1 promoter, URA3 marker
CEN, GPD1 promoter, URA3 marker
2μ, ADH1 promoter, URA3 marker
2μ, TEF1 promoter, URA3 marker,
p416 ADH with Kar2 and Aβ42 sequence
p416 TEF with Kar2 and Aβ42 sequence
p426 ADH with Kar2 and Aβ42 sequence
p416 GPD with Kar2 and Aβ42 sequence
p426 TEF with Kar2 and Aβ42 sequence
p416 GPD with Kar2 and Aβ40 sequence
Mumberg et al. (1995)
Mumberg et al. (1995)
Mumberg et al. (1995)
Mumberg et al. (1995)
Mumberg et al. (1995)
This study
This study
This study
This study
This study
This study
MATα his31 ura3–52 MAL2–8c SUC2
CEN.PK 113–11C/p416 GPD
CEN.PK 113–11C/p426 ADH
CEN.PK 113–11C/p416 ADH-Kar2-Aβ42
CEN.PK 113–11C/p416 TEF-Kar2-Aβ42
CEN.PK 113–11C/p426 ADH-Kar2-Aβ42
CEN.PK 113–11C/p416 GPD-Kar2-Aβ42
CEN.PK 113–11C/p426 TEF-Kar2-Aβ42
CEN.PK 113–11C/p416 GPD-Kar2-Aβ40
This study
This study
This study
This study
This study
This study
This study
This study
This study
Aβ can also accumulate within the cells, which may contribute
to disease onset or progression (LaFerla, Green and Oddo 2007). It
has been proposed that senile plaques originated from intraneuronal Aβ as a result of its release after neuronal death (Gouras
et al. 2000). Intracellular Aβ has been pointed out to be involved
in early stages of the pathogenesis, directly causing neurotoxicity and initiating AD (Takahashi et al. 2002; Knobloch et al. 2007).
There is growing evidence suggesting that alterations in energy metabolism are among the earliest events that occur in the
AD-affected brain (Fukui and Moraes 2008). Mitochondria produce most of the ATP, and they are key regulators of cell survival
and cell death, hence playing a central role in aging and AD onset
and progression. Increasing evidence shows the occurrence of
impaired mitochondrial functions in AD patients and in different models, such as impaired electron transfer, ATP synthesis,
mitochondrial transcription, mitochondrial pre-protein maturation and protein synthesis, upregulation of voltage-dependent
anion channel, decreased mitochondrial membrane potential,
as well as increased production of reactive oxygen species (ROS)
(Aleardi et al. 2005; Matsumoto et al. 2006; Crouch et al. 2008; Yao
et al. 2009; Manczak et al. 2010; Calkins et al. 2011; Mossmann
et al. 2014). Nevertheless, the precise mechanism of mitochondrial dysfunction in AD pathogenesis still remains uncertain.
Although different biological model systems have been used
to study the role of Aβ, the complexity of AD has still prevented
a comprehensive understanding of Aβ toxicity (Link 1995; Magrane et al. 2004; Crowther et al. 2005). Yeast Saccharomyces cerevisiae is a single cell eukarya organism that can be used as a
model to study conserved biological processes, but it can also
be used to study human proteins in so-called humanized yeast
models which can be a valuable tool to study intracellular toxicity of Aβ. This model system has excellent genetic and molecular tools that can be used for molecular and cell biology, systems
biology, single cell methods and also for screening chemical libraries that might modulate amyloid toxicity (Petranovic and
Nielsen 2008; Khurana and Lindquist 2010; Munoz et al. 2012;
Mirisola, Braun and Petranovic 2014). During the last decade,
S. cerevisiae has been exploited to study toxicity, aggregation
and localization of Aβ or as screening tool for identification of
compounds influencing Aβ oligomerization (Zhang et al. 1997;
Komano et al. 1998; Caine et al. 2007; Macreadie et al. 2008; Winderickx et al. 2008). However, in these previous studies, Aβ was
expressed in the cytosol and recent studies in yeast showed
that the secretory pathway is important for the generation of
Aβ toxic species (Treusch et al. 2011; D’Angelo et al. 2013). The
completely unbiased screen of compounds was performed for
rescue of Aβ toxicity (Matlack et al. 2014). Additionally, the previous models used either inducible expression systems (Treusch
et al. 2011) or non-native versions of Aβ peptide (D’Angelo et al.
2013).
Here we describe a yeast model of Aβ cellular toxicity that
expresses native Aβ-peptides (Aβ40 and Aβ42), which are constitutively expressed, and possess the endoplasmic reticulum
(ER) signal sequence for secretion. We demonstrate that this system produced Aβ40 and Aβ42 monomers and oligomers and induced cytotoxicity in yeast. Additionally, we found a decrease
in the mitochondrial functions which was accompanied by an
elevated production of ROS and reduced proteasomal activities.
This model could also be developed as a discovery platform for
screening of compounds that revert Aβ toxicity.
MATERIALS AND METHODS
Yeast strains, plasmids and standard growth conditions
All yeast strains used in this work are derivatives of S. cerevisiae
CEN.PK 113–11C (MATα his31 ura3–52 MAL2–8c SUC2). Plasmid
constructions were performed following standard molecular biology techniques using Escherichia coli DH5α stain (fhuA2 (argFlacZ) U169 phoA glnV44 80 (lacZ)M15 gyrA96 recA1 relA1 endA1
thi-1 hsdR17).
The different yeast shuttle plasmids were used for the expression of Aβ in yeast (listed in Table 1). The ER targeting
signal sequence from Kar2 and Aβ42/ Aβ40 sequences were
Chen and Petranovic
amplified by PCR from pDONR221-Aβ42 and pDONR221-Aβ40
plasmids (kindly provided by Prof. Susan Lindquist, Whitehead
Institute for Biomedical Research at MIT). The yeast transformations were performed using a standard lithium acetate method
and selected on synthetic dextrose plates SD-Ura− . For cultivation, all stains were grown at 30◦ C either in liquid SD-Ura− media
(0.067% yeast nitrogen base, 2% glucose excluding uracil) or Delft
medium (0.5% (NH4 )2 SO4 , 0.3% of KH2 PO4 , 0.05% of MgSO4 ·7H2 O,
0.1% vitamin solution and 0.1% trace metal solution, 2% ethanol,
pH 6.5) as described previously (Chen et al. 2013).
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cubated with 0.5 μg ml−1 PI (Invitrogen) for 20 min, washed with
PBS and then cells positive for red PI staining were observed under the fluorescence microscope and counted on a flow cytometer (Guava System). A total of 5000 cells for each sample were
counted and the percentage of cells that stained positive for PI
were recorded. The experiments were done in biological triplicates. As a positive control, we used cells that were incubated at
95◦ C for 20 min. Statistical significance was determined by using
the two-tailed Student’s t-test.
Protein extraction and western blotting
Batch fermentations
Respiration assays were carried out in the DasGip 1.0-liter
stirrer-pro bioreactors (DASGIP GmbH, Germany) with a 0.6-liter
working volume under aerobic conditions. The temperature was
controlled at 30◦ C with agitation at 600 rpm and aeration at
1 vvm. Dissolved oxygen was controlled above 30% throughout the experiment. The pH was maintained at 5.0 with 2 M
KOH, in response to the pH sensor (Mettler Toledo, Switzerland).
The emission of CO2 and residual O2 was monitored by DasGip
fedbatch pro gas analysis systems. Strains were grown in Delft
medium. All fermentations were performed in biological duplicates.
The supernatants filtered through 0.45 μm membrane were
loaded to an Aminex HPX-87H column (Bio Rad, Hercules, USA)
on a Summit HPLC (Dionex, Sunnyvale, CA, USA) to measure the
concentration of ethanol in the cultures. The samples were running with a flow rate of 0.5 ml min−1 at 65◦ C using 0.5 mM H2 SO4
as the mobile phase.
Strains were grown to post-diauxic phase in SD-Ura− medium
at 30◦ C, and cells were spun down at 2000 g for 5 min. Cell
pellets were resuspended in 200 μl of cold lysis buffer (50 mM
HEPES pH7.5, 150 mM NaCI, 2.5 mM EDTA, 1% v/v Triton X-100)
with freshly added Complete Mini Protease Inhibitors (Roche).
Then the 200 μl of glass beads was added and the cells were
shaken for 3 min on maximum speed. Following centrifugation at 13 000 g for 15 min, the supernatant was collected and
protein concentrations were measured using Micro BCA protein
assay kit (Thermo Scientific). A total of 50 μg protein of each
sample was separated by a 4–12% Bis-Tris gel (invitrogen). Proteins were electrically transferred to 0.2 micron PVDF membrane
(BioRad) using a Bio-Rad semi-dry transfer system. The membranes were probed with monoclonal mouse anti-Aβ antibody (6E10, 1:2000, Covance) or anti-GAPDH (1:2000, Santa Cruz)
overnight at 4◦ C. Binding was detected with the ECL Prime
reagent (GE Healthcare) and the ChemiDoc XRS image analyzer
(Bio Rad).
Spotting assay
Immunostaining of intracellular Aβ
All spotting assays were performed under the same conditions.
Tenfold serial dilution in sterile water starting with an equal
number of cells (0.2 OD600 ) was used. Spotting assays were done
in biological triplicates. Drops of 4.5 μl were plated onto the SDUra− plates. Plates were incubated at 30◦ C for 2–3 days.
Strains were cultured to post-diauxic phase in SD-Ura− medium.
Cells were harvested and fixed in 5 ml of 50 mM KPO4 (pH6.5),
1 mM MgCI2 and 4% formaldehyde for 2 h. After fixation, the
cells were washed three times in 5 ml of PM (0.1 M KPO4 pH
7.5, 1 mM MgCI2 ) and then resuspended in PMST (0.1 M KPO4 pH
7.5, 1 mM MgCI2 , 1 M Sorbitol, 0.1% Triton X-100) to a final OD600
of 10. A total of 100 μl of cells were incubated with 0.6 μl of 2mercaptoethanol and 1 mg ml−1 zymolyase (Zymo Research) for
20 min. Spheroplasted cells were washed three times in PMST
and attached to a polylysine-coated coverslips. The adherent
cells were blocked in 0.5% BSA/PMST for 30 min, and incubated
overnight at 4◦ C with primary antibody (1:200 6E10 Aβ antibody)
diluted in 0.5% BSA/PMST. Cells were washed in PMST, incubated with secondary antibody (1:300 anti-mouse Alexa 488) for
2 h at RT and incubated with 0.4 mg ml−1 DAPI (4’,6-diamidino2-phenylindole) for 5 min. Cells were mounted in Vectashield
(Vector Laboratories). The images were acquired using a Leica AF 6000 inverted microscope, and processed with the Leica
Application Suite (LAS) software.
Chronological life span assay
Chronological life span (CLS) was measured as previously described (Fabrizio and Longo 2003). Strains grown overnight at
30◦ C in SD-Ura− medium were diluted into 20 ml of medium to
give an initial OD600 of 0.1. Cultures were grown with shaking
(200 rpm) at 30◦ C to ensure proper aeration. After 24 h, these
cultures had reached stationary phase, and this time point was
designated day 1. Every 2 days, aliquots from the culture were serially diluted in YPD to achieve a cell density of ∼ 4 × 103 ml−1 .
One hundred microliters of the dilution was plated on to YPD
plates, and incubated at 30◦ C for 2 days. The viability was estimated by colony forming units (CFUs). Viability at day 2 was considered to be the initial survival (100%). Averages and standard
deviations for at least three biological replicates were calculated
for each experiment.
Propidium iodide staining
Propidium iodide (PI) is a vital stain which stains cells with disintegrated plasma membranes, which are from a morphologically based state of view dead cells. For the PI staining of dead
yeast cells, strains were pre-grown overnight and then diluted to
0.1 OD600 in 20 ml of SD-Ura− medium. A total of 0.5 OD600 unit
of cells were taken at day 1, 3 and 9 of incubation. Cells were in-
Measurements of ROS
The production of ROS was measured using dihydrorhodamine
123 (DHR, Sigma Aldrich). Strains were grown aerobically at
30◦ C in Delft medium. Samples of cells were grown to midexponential phase. A total of 0.5 OD600 of cells were harvested and incubated with 5 μM DHR for 20 min and used
for analysis under the fluorescence microscope and for the
flow cytometer analysis (Guava System). In each sample, 5000
cells were counted. The experiments were done in biological
duplicates.
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FEMS Yeast Research, 2015, Vol. 15, No. 6
Figure 1. Construction of yeast strains expressing constitutively Aβ 42 targeted for secretion. (A) Schematic map of the plasmids, different promoters (shaded boxes)
and the Kar2-Aβ 42 gene (striped box) used for the Aβ 42 expression. Numbers below the boxes represent the regions of the promoters relative to the start codon.
(B) Spot test (10-fold dilutions) to measure the relative viability of control and Aβ 42 strains in exponential phase.
Proteasome activity assay
The 20S proteasomal activity was assessed using ProteasomeGlo cell-based assay (Promega GmbH, Germany) according
to the manufacturer’s instructions. Strains were grown aerobically at 30◦ C in Delft medium and harvested at OD600
of 1.0. Different numbers (20 000, 40 000 and 80 000) of
cells were mixed with specific luminogenic proteasome
substrates (Suc-LLVY-aminoluciferin for chymotrypsin-like
activity, Z-LRR-aminoluciferin for trypsin-like activity and
Z-nLPnLD-aminoluciferin for caspase-like activity), and substrate luminescence was measured by the FLUOstar Galaxy
plate reader from BMG labtechnologies. All measurements were
made in triplicate. In order to exclude that the detected signal
resulted from non-proteasome cleavage of the aminoluciferin
substrates by other proteases, some samples were incubated
with the proteasome inhibitor epoxomicin for 1 h before
measurement in a previous experiment. Since no significant
luminescence was detected in the presence of epoxomicin, the
signals detected in the samples without epoxomicin could be
completely attributed to the activity of the 20S proteasome.
RESULTS
Effect of Aβ40 and Aβ42 on growth, CLS and cell death
A series of vectors were used for constitutive Aβ42 expression at
different levels. We chose three different promoters (ADH1: alcohol dehydrogenase 1; TEF1: translation elongation factor 1α and
GPD1: glyceraldehyde-3-phosphate dehydrogenase) in two different plasmids (centromeric plasmid with a low copy number
(CEN) and a high copy number plasmid (2μ)). Both plasmids are
carrying the URA3 selective marker. The CEN derivatives were
named p416 series, and the 2μ derivatives p426 (see Table 1 and
Fig. 1A for plasmids and strains). The strength of transcription
from different promoters is ADH1 < TEF1 < GPD1. The expression
levels of these vectors were measured previously by cloning the
lacZ gene downstream of the promoters and the β-galactosidase
activity was determined: p416 ADH (3.0 units), p416 TEF (47.9
units), p426 ADH (90.8 units), p416 GPD (215 units) and p426 TEF
(508 units) (Mumberg, Muller and Funk 1995).
The constructed yeast strains with different Aβ42 expression
levels were first tested for growth on plates (Fig. 1B) and in liquid SD-Ura− medium (Fig. 2A, Table 2). Compared to the controls
(empty vector p416 EP), Aβ42 expressed in p416 ADH and p416
TEF plasmids did not affect growth (Table 2). Aβ42 expressed in
p416 GPD plasmid caused an 11.3% decrease in growth (t-test,
P < 0.05). Growth of p426 ADH-Aβ42 was reduced by 12.1%
(t-test, P < 0.05), when compared to the control (high copy number empty vector p426 EP). The growth of cells expressing p416
GPD-Aβ42 and p426 ADH-Aβ42 was similar; however, the standard deviation of growth rate in high copy number plasmids
was higher (0.013) than in low copy number plasmids (0.004). To
get the stable expression of Aβ42, the p416 GPD plasmid was
used for the experiments. This construct is a centromeric plasmid stably maintained at one or two copies per cell, and has
the strong constitutive GPD promoter for heterologous gene expression. Aβ42 was also expressed in p426 TEF plasmid, but the
strain did not grow. This hints to a strong cytotoxic effect of Aβ42
at high expression level, so this strain was not used for further
studies.
The expression of Aβ42 from p416 GPD plasmid caused a
small but statistically significant reduction in growth (reduction
of 11.3%, P < 0.05, Table 2). Expression of Aβ40 (also cloned in
p416 GPD plasmid) was less toxic when compared to control (reduction of 2.3%) (Fig. 2A). Aβ40 is also an APP cleavage product but it was found to be less cytotoxic in various AD models
(Luheshi et al. 2007) presumably because of lesser tendency to
aggregate when compared to Aβ42 peptides.
To measure the long-term effect of Aβ42 and Aβ40 expression on the cells, we also examined the CLS. The number of viable cells in stationary phase of the control (empty vector p416
EP), the Aβ42 and the Aβ40 strains were determined over the
course of several days using either CFUs or by spotting assay
(Fig. 2B and C, respectively). Compared to control, Aβ42 strain
displayed a drastically reduced ability to maintain viability in
stationary phase and had therefore the CLS shortened from 13
days to 9 days. Though the expression of Aβ40 did not affect
growth in exponential phase (Fig. 2A), it did reduce the CLS to 11
days.
We also performed a PI staining to detect dead cells. PI is a vital stain that cannot permeate into living cells but which stains
Chen and Petranovic
5
Figure 2. Aβ expression reduced growth and CLS. The p416 GPD plasmid was used to express the Aβ 40 and Aβ 42 proteins. The CEN.PK.113–11C cells were transformed
with an empty vector (control), Aβ 40 or Aβ 42 plasmids. (A) Growth rate of control, Aβ 40 and Aβ 42 strains. (B) CLS assays of control, Aβ 40 and Aβ 42 strains. (C) Relative
viability of control, Aβ 40 and Aβ 42 strains by plating serial 10-fold dilutions from CLS day 1 and day 9. (D) Flow cytometry analysis of PI staining following the CLS.
The asterisk (∗ ) indicates a value of P < 0.05. (E) Representative micrographs of PI staining of cells from CLS on day 3. All results are representative of at least three
independent experiments and error bar indicates SD. The scale bars are 10 μm.
Table 2. Growth of CEN.PK.113–11C cells transformed with an empty
vector (control) or Aβ42 in different vectors.
p416 EP
p416 ADH-Aβ42
p416 TEF-Aβ42
p416 GPD-Aβ42
p426 EP
p426 ADH-Aβ42
Generation time (min)
Growth rate (h−1 )
108.83 ± 1.89
99.86 ± 1.62
102.54 ± 2.29
122.69 ± 1.54
117.69 ± 5.59
133.93 ± 5.74
0.382 ± 0.0066
0.416 ± 0.011
0.406 ± 0.009
0.339 ± 0.004
0.354 ± 0.017
0.311 ± 0.013
cells with damaged plasma membrane. We took samples at different days including during late stationary phase. This revealed
that expression of Aβ42 increased cell death from day 1 (Fig. 2D
and E).
Expression and localization of Aβ40 and Aβ42
The expression of Aβ peptides was detected by western blot
analysis using the Aβ-specific antibody, 6E10. The strains produced a peptide of the expected size (4 kDa). When lysates were
loaded without denaturation by boiling in the denaturing loading buffer, we detected higher molecular weight species of Aβ,
corresponding to different oligomers. In the Aβ42 strain, more
oligomers were formed, and high molecular weight oligomers
were detectable (Fig. 3A). Trimers and tetramers that are detectable in the Aβ42 strain (but not in the Aβ40 strain) are resolved when the sample is denatured by boiling in the SDS
buffer, prior to SDS-PAGE. Thus, oligomeric Aβ forms seem to
contribute to toxicity in yeast, as they probably do in neurons.
In vivo cellular localization of Aβ peptides was confirmed by
immunostaining. In the Aβ42 strain, Aβ was detected along the
ER and was distributed in small foci throughout the cell. The ER
was shown as a ring surrounding the nucleus which was stained
with DAPI. By contrast, in the less toxic Aβ40 strain Aβ peptides
seem to be more dispersed in the cell (Fig. 3B).
Aβ42 affects mitochondrial respiratory capacity and
induces production of ROS
Mitochondrial dysfunction seems to play a key role in AD.
We therefore tested whether the toxicity triggered by the expression of Aβ42 in yeast was associated with mitochondrial
dysfunction in our model. We measured the carbon dioxide
transfer rate (CTR), during respiration phase, as an indicator of
respiratory rates. The cells were grown in the batch fermentation mode in the controlled bioreactor, in defined medium. In
order to test the strains during the respiration, we used a nonfermentable carbon source, ethanol. Under these conditions, the
CTR of the control, Aβ40 and Aβ42 strains was similar and unchanged during early exponential growth. By contrast, the respiratory rate in the Aβ42 strain was significantly decreased from
mid-exponential phase (to 40% compared to control). The respiratory rate of Aβ40 strain decreased to a much lesser extent
(Fig. 4B and C). The biomass yield was calculated based on the
ratio of biomass produced, and ethanol consumed during the
respiration. The biomass yield decreased in the Aβ42 strain after
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FEMS Yeast Research, 2015, Vol. 15, No. 6
Figure 3. Oligomerization of Aβ . The Aβ 40 and Aβ 42 peptides were expressed in p416 GPD plasmid. (A) Western blot analysis of Aβ 40 and Aβ 42 strains, using
the Aβ -specific 6E10 antibody. GAPDH was used as a loading control. Samples were loaded onto a Bis-Tris gel with and without prior boiling in SDS sample buffer.
(B) Immunostaining analysis of Aβ 40 and Aβ 42 localization using the 6E10 Aβ -specific antibody in vivo. Nuclei were visualized by DAPI staining. The scale bars are 5
μm.
the mid-exponential phase (to 65% when compared to control)
(Fig. 4D). This result correlates well with the slow growth phenotype of Aβ42 strain under respiratory conditions (Fig. 4A).
Besides producing ATP, mitochondria are also involved in initiation of cell death and are also the main source of cellular ROS,
which can damage cellular components and biomolecules and
thus are involved in the pathogenesis of AD (Shahul and GingYuek 2011). We measured intracellular ROS levels by flow cytometry using the non-fluorescent dihydrorhodamine 123 (DHR), to
which yeast cells are permeable. DHR can be oxidized by the
intracellular ROS into rhodamine 123, a fluorescent compound
to which cells are impermeable, and which can be detected by
flow cytometry. During respiration Aβ42 strain exhibited significantly higher levels of ROS, when compared to the control and
the Aβ40 strain (Fig. 4E). We also qualitatively confirmed these
results using fluorescence microscopy (Fig. 4F).
Aβ42 affects the proteasomal activity
The ubiquitin-proteasome system (UPS) is central for the degradation of abnormal proteins such as misfolded and oxidized
proteins, and for the maintenance of cellular proteostasis. We
therefore measured all three proteolytic activities of the 20S
proteasome (the chymotrypsin-, caspase- and trypsin-like proteases) in exponentially growing cells. All three proteolytic activities were lower in Aβ42 strain compare to control and Aβ40
strains (Fig. 5). This suggests that an excess of misfolded proteins in Aβ42 strain inhibits the proteasome to degrade other
substrates which possibly also contributes to the cytotoxic phenotype.
DISCUSSION
Yeast has been used as a model for several neurological disorders characterized by protein misfolding and aggregation (Winderickx et al. 2008; Braun et al. 2010; Khurana and Lindquist 2010).
In order to study Aβ cytotoxicity, a few approaches have been
used previously including chemically synthesized Aβ, intracellular fused Aβ and intracellular recombinant forms. Studies in Candida glabrata showed that the chemically synthesized
Aβ induced oligomerization-dependent cytotoxicity (Bharadwaj
et al. 2008). The in vivo oligomerization of Aβ was studied using Aβ/Sup35p fusion expressed in yeast. The yeast translation termination factor Sup35p is essentially composed of three
domains: the N-terminal domain (N), middle domain (M) and
C-terminal release factor domain (RF). The Aβ peptide was fused
to the MRF domain of Sup35p, thereby causing oligomerization
of MRF and growth reduction (Bagriantsev and Liebman 2006).
Similarly, when the Aβ peptide was fused to the green fluorescent protein (GFP) at the C- or N-terminus, it was shown that
the heat shock response was induced and there was reduction
of cell growth (Caine et al. 2007). While Aβ fusions are helpful in
many cases, it can be argued that fusions could alter the properties of Aβ, e.g. the intracellular Aβ could be stabilized and thus
less toxic, so a system with native Aβ could be useful.
Previous studies showed that there was no detectable native
Aβ expression in yeast, despite the use of a copper-inducible
expression system that has previously proved suitable for the
production of toxic proteins such as HIV-1 Vpr (Macreadie et al.
1995). In these previous studies, Aβ was expressed in the cytosol, whereas native Aβ derives from the APP, which generates Aβ peptide within the secretory and endosomal pathways.
APP processing primarily occurs in the secretory network, with
the release of Aβ into extracellular space, and subsequently
internalized and recycled through endosomal vesicles and the
trans-Golgi network via endocytosis (Thinakaran and Koo 2008).
Recently, a new screen based on a secreted form of Aβ in yeast
revealed the importance of the endocytic pathway in cellular
toxicity (Treusch et al. 2011). This finding was confirmed by other
yeast Aβ expression models (D’Angelo et al. 2013). The GFP-Aβ
fusion targeted to the secretory pathway indicated decreased
growth rates and markedly reduction of oxygen consumption on
obligatory respiratory growth media. This yeast model of Aβ toxicity was used for a completely unbiased screen of compounds
Chen and Petranovic
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Figure 4. The effect of Aβ expression on mitochondrial respiration and ROS production. The Aβ 40 and Aβ 42 peptides were expressed in p416 GPD plasmid. The strains
were grown aerobically in batch fermentations in the Delft medium. (A) Growth of Aβ 40 and Aβ 42 strains during respiration. (B) Analysis of CTR during respiration.
(C) The comparison of respiratory rates in mid-exponential phase. (D) The comparison of biomass yield in mid-exponential phase. (E) Flow cytometry analysis of ROS
accumulation using DHR 123 staining in mid-exponential phase. Results are mean ± SD of at least two measurements performed on two independent cell cultures.
The asterisk (∗ ) indicates a value of P < 0.05 according to Student’s t test. (F) Visualization of ROS producing cells in mid-exponential phase using DHR 123. The scale
bars are 10 μm.
for rescue of Aβ toxicity. A small number of cytoprotective compounds including 8-hydroxyquinolines were identified (Matlack
et al. 2014).
In this study, native Aβ was fused to an ER targeting signal sequence to direct correctly Aβ into the secretory compartments. Different plasmids and promoters were tested to generate different levels of Aβ42 peptides in yeast. We selected
the strain exhibiting intermediate levels of toxicity that allowed
for the analysis of Aβ42-induced cellular toxicity. The Aβ42
peptide expressed constitutively formed oligomers and aggregates throughout the cells, seemingly also along the secretory
network. The Aβ42 strain showed a reduced growth rate, in-
creased number of dead cells and significantly shorter chronological life span. Therefore, it is concluded that the expression of
Aβ42 and the formation of oligomers impose a cytotoxic stress.
The Aβ42 strain showed also impaired respiration capacity.
Cells producing Aβ42 showed significant reduction of respiratory rate and biomass yield from mid-exponential stage, which
was accompanied by an elevated production of ROS. Accumulating data suggest that mitochondrial dysfunction and oxidative
stress occur in the neurons as well as in peripheral tissues of
AD patients (Fukui and Moraes 2008). Studies have shown that
Aβ is accumulating in mitochondria in post-mortem AD brain
samples, in patients with cortical plaques and in TgAPP mice
8
FEMS Yeast Research, 2015, Vol. 15, No. 6
Figure 5. The effect of Aβ expression on proteasomal activity. Proteasomal activity of the chymotrypsin-, caspase- and trypsin-like proteolytic activities of the 20S
proteasome were measured as luminescence in control, Aβ 40 and Aβ 42 strains. The luminescent signal was proportional to the amount of proteasome activity in
cells and recorded as relative luminescent units (RLU). Data are represented as mean ± SD from three independent experiments.
(Lustbader et al. 2004; Caspersen et al. 2005; Petersen et al. 2008).
In TgAPP mice, mitochondrial Aβ accumulation occurs prior to
plaque formation, indicating that this is an early event in the
development of pathogenesis. The mitochondria isolated from
S. cerevisiae strains, mouse and human brain tissues shows signs
of mitochondrial impairment upon prolonged exposure to cytosolic Aβ42, including inhibition of mitochondrial pre-protein
maturation, increased ROS production, decreased mitochondrial
membrane potential and reduced oxygen consumption (Mossmann et al. 2014).
It is interesting to consider how Aβ could reach the mitochondria. Intracellular Aβ42 has been shown to accumulate in
intracellular multivesicular bodies (Gouras et al. 2000) and it is
possible that Aβ leaking from these vesicles could reach the
mitochondria. Hansson et al. showed that Aβ ‘fed’ extracellularly is internalized by human neuroblastoma cells and later
partly localized to mitochondria (Petersen et al. 2008). These data
suggest that secreted Aβ can be re-internalized into cells and
reach mitochondria. We found previously that yeast can uptake considerable amounts of secreted protein (in the range of
1 g L−1 day−1 ). Characterizing the physiological state and combining metabolomics and transcriptomics, we have previously
identified metabolic and regulatory markers that were consistent with uptake of whole proteins by endocytosis, followed by
intracellular degradation and catabolism of substituent amino
acids (Tyo et al. 2014).
Dysfunctions of the mitochondria and UPS have been
strongly associated with the pathogenesis of many neurodegenerative disorders, including AD (Segref et al. 2014). However, the
order in which these events occur as well as the intracellular
mechanisms remain unclear. Recent study shows that the accumulation of UBB+1 , a frameshift variant of ubiquitin B, induces enhancement of the basic amino acids arginine, ornithine
and lysine at mitochondria as a decisive toxic event (Braun
et al. 2015). In our model, the proteolytic activities were measured and showed decrease in Aβ42 strain compare to control and Aβ40 strains. A recent study has shown that the acute
mitochondrial stress results in proteasomes dissociation, mitochondrial networks fragment and cellular ROS levels increase
(Livnat-Levanon et al. 2014). Conversely, the proteasome inhibition leads to mitochondrial oxidation followed by cytosolic oxidation, which can be prevented by a mitochondrial-targeted antioxidant (Maharjan et al. 2014).
In this study, we have created a yeast model for expression
of native Aβ peptides that are constitutively expressed, form
intracellular oligomers, are targeted to the secretory pathway
and induce cytotoxic effects. We have also found that the toxicity is influencing the mitochondria, respiration and the proteasomal system. We thus believe that this model will be used
in the future for discovery of mechanism of Aβ42 toxicity.
ACKNOWLEDGEMENTS
We are grateful to Professor Susan Lindquist for giving us the
inital Aβ plasmids and to Dr Yun Chen and Dr Mingtao Huang
for their assistance with fermentations.
FUNDING
This work was supported by grants from the Novo Nordisk Foundation, Kristina Stenborgs Foundation and Sven & Lilly Lawski
Foundation.
Conflict of interest. None declared.
REFERENCES
Aleardi AM, Benard G, Augereau O, et al. Gradual alteration of
mitochondrial structure and function by beta-amyloids: importance of membrane viscosity changes, energy deprivation, reactive oxygen species production, and cytochrome c
release. J Bioenerg Biomembr 2005;37:207–25.
Bagriantsev S, Liebman S. Modulation of Abeta42 low-n
oligomerization using a novel yeast reporter system. BMC Biol
2006;4:32.
Bharadwaj P, Waddington L, Varghese J, et al. A new method
to measure cellular toxicity of non-fibrillar and fibrillar
Alzheimer’s A beta using yeast. J Alzheimers Dis 2008;13:147–
50.
Braun RJ, Buttner S, Ring J, et al. Nervous yeast: modeling neurotoxic cell death. Trends Biochem Sci 2010;35:135–44.
Braun RJ, Sommer C, Leibiger C, et al. Accumulation of basic
amino acids at mitochondria dictates the cytotoxicity of
aberrant ubiquitin. Cell Rep 2015;10:1557–71.
Caine J, Sankovich S, Antony H, et al. Alzheimer’s A beta fused to
green fluorescent protein induces growth stress and a heat
shock response. FEMS Yeast Res 2007;7:1230–6.
Calkins MJ, Manczak M, Mao PZ, et al. Impaired mitochondrial
biogenesis, defective axonal transport of mitochondria, abnormal mitochondrial dynamics and synaptic degeneration
Chen and Petranovic
in a mouse model of Alzheimer’s disease. Hum Mol Genet
2011;20:4515–29.
Caspersen C, Wang N, Yao J, et al. Mitochondrial A beta: a potential focal point for neuronal metabolic dysfunction in
Alzheimer’s disease. FASEB J 2005;19:2040–1.
Chen Y, Daviet L, Schalk M, et al. Establishing a platform cell
factory through engineering of yeast acetyl-CoA metabolism.
Metab Eng 2013;15:48–54.
Crouch PJ, Harding S-ME, White AR, et al. Mechanisms of Aβ
mediated neurodegeneration in Alzheimer’s disease. Int J
Biochem Cell B 2008;40:181–98.
Crowther DC, Kinghorn KJ, Miranda E, et al. Intraneuronal
Aβ, non-amyloid aggregates and neurodegeneration in
a Drosophila model of Alzheimer’s disease. Neuroscience
2005;132:123–35.
D’Angelo F, Vignaud H, Di Martino J, et al. A yeast model
for amyloid-β aggregation exemplifies the role of membrane trafficking and PICALM in cytotoxicity. Dis Model Mech
2013;6:206–16.
Fabrizio P, Longo VD. The chronological life span of Saccharomyces cerevisiae. Aging Cell 2003;2:73–81.
Ferri CP, Prince M, Brayne C, et al. Global prevalence of dementia:
a Delphi consensus study. Lancet 2005;366:2112–7.
Fukui H, Moraes CT. The mitochondrial impairment, oxidative stress and neurodegeneration connection: reality
or just an attractive hypothesis? Trends Neurosci 2008;31:
251–6.
Gouras GK, Tsai J, Naslund J, et al. Intraneuronal Aβ42 accumulation in human brain. Am J Pathol 2000;156:15–20.
Hardy J, Selkoe DJ. Medicine—the amyloid hypothesis of
Alzheimer’s disease: progress and problems on the road to
therapeutics. Science 2002;297:353–6.
Hartley DM, Walsh DM, Ye CPP, et al. Protofibrillar intermediates of amyloid beta-protein induce acute electrophysiological changes and progressive neurotoxicity in cortical neurons. J Neurosci 1999;19:8876–84.
Khurana V, Lindquist S. Modelling neurodegeneration in Saccharomyces cerevisiae: why cook with baker’s yeast? Nat Rev Neurosci 2010;11:436–49.
Knobloch M, Konietzko U, Krebs DC, et al. Intracellular Aβ and
cognitive deficits precede β-amyloid deposition in transgenic
arcAβ mice. Neurobiol Aging 2007;28:1297–306.
Komano H, Seeger M, Gandy S, et al. Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases
in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in
yeast. J Biol Chem 1998;273:31648–51.
LaFerla FM, Green KN, Oddo S. Intracellular amyloid-beta in
Alzheimer’s disease. Nat Rev Neurosci 2007;8:499–509.
Link CD. Expression of human beta-amyloid peptide in
transgenic Caenorhabditis elegans. P Natl Acad Sci USA
1995;92:9368–72.
Livnat-Levanon N, Kevei E, Kleifeld O, et al. Reversible 26S proteasome disassembly upon mitochondrial stress. Cell Rep
2014;7:1371–80.
Luheshi LM, Tartaglia GG, Brorsson AC, et al. Systematic in
vivo analysis of the intrinsic determinants of amyloid beta
pathogenicity. PLoS Biol 2007;5:2493–500.
Lustbader JW, Cirilli M, Lin C, et al. ABAD directly links A
beta to mitochondrial toxicity in Alzheimer’s disease. Science
2004;304:448–52.
McLean CA, Cherny RA, Fraser FW, et al. Soluble pool of Aβ amyloid as a determinant of severity of neurodegeneration in
Alzheimer’s disease. Ann Neurol 1999;46:860–6.
9
Macreadie I, Lotfi-Miri M, Mohotti S, et al. Validation of folate in a
convenient yeast assay suited for identification of inhibitors
of Alzheimer’s amyloid-beta Aggregation. J Alzheimers Dis
2008;15:391–6.
Macreadie IG, Castelli LA, Hewish DR, et al. A domain of human immunodeficiency virus type 1 Vpr containing repeated H(S/F)RIG amino acid motifs causes cell growth arrest and structural defects. P Natl Acad Sci USA 1995;92:
2770–4.
Magrane J, Smith RC, Walsh K, et al. Heat shock protein 70
participates in the neuroprotective response to intracellularly expressed beta-amyloid in neurons. J Neurosci 2004;24:
1700–6.
Maharjan S, Oku M, Tsuda M, et al. Mitochondrial impairment
triggers cytosolic oxidative stress and cell death following
proteasome inhibition. Sci Rep 2014;4:11.
Manczak M, Mao P, Calkins M, et al. Mitochondriatargeted antioxidants protect against Abeta toxicity in
Alzheimer’s disease neurons. J Alzheimers Dis 2010;20:
S609–S31.
Matlack KES, Tardiff DF, Narayan P, et al. Clioquinol promotes the
degradation of metal-dependent amyloid-β (Aβ) oligomers to
restore endocytosis and ameliorate Aβ toxicity. P Natl Acad
Sci USA 2014;111:4013–8.
Matsumoto K, Akao Y, Yi H, et al. Overexpression of amyloid
precursor protein induces susceptibility to oxidative stress
in human neuroblastoma SH-SY5Y cells. J Neural Transm
2006;113:125–35.
Mirisola MG, Braun RJ, Petranovic D. Approaches to study
yeast cell aging and death. FEMS Yeast Res 2014;14:
109–18.
Mossmann D, Vögtle FN, Taskin Asli A, et al. Amyloid-β peptide
induces mitochondrial dysfunction by inhibition of preprotein maturation. Cell Metab 2014;20:662–9.
Mumberg D, Muller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 1995;156:119–22.
Munoz AJ, Wanichthanarak K, Meza E, et al. Systems biology of yeast cell death. FEMS Yeast Res 2012;12:
249–65.
Petersen CAH, Alikhani N, Behbahani H, et al. The amyloid betapeptide is imported into mitochondria via the TOM import
machinery and localized to mitochondrial cristae. P Natl Acad
Sci USA 2008;105:13145–50.
Petranovic D, Nielsen J. Can yeast systems biology contribute
to the understanding of human disease? Trends Biotechnol
2008;26:584–90.
Segref A, Kevei E, Pokrzywa W, et al. Pathogenesis of human mitochondrial diseases is modulated by reduced activity of the ubiquitin/proteasome system. Cell Metab 2014;19:
642–52.
Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy.
Physiol Rev 2001;81:741–66.
Selkoe DJ, Wolfe MS. Presenilin: running with scissors in the
membrane. Cell 2007;131:215–21.
Shahul H, Ging-Yuek RH. The role of mitochondria in aging, neurodegenerative disease, and future therapeutic options. B C
Med J 2011;53:188–92.
Takahashi RH, Milner TA, Li F, et al. Intraneuronal Alzheimer
Aβ42 Accumulates in multivesicular bodies and is associated with synaptic pathology. Am J Pathol 2002;161:
1869–79.
Thinakaran G, Koo EH. Amyloid precursor protein trafficking,
processing, and function. J Biol Chem 2008;283:29615–9.
10
FEMS Yeast Research, 2015, Vol. 15, No. 6
Treusch S, Hamamichi S, Goodman JL, et al. Functional
links between Aβ toxicity, endocytic trafficking and
Alzheimer’s disease risk factors in yeast. Science 2011;334:
1241–5.
Tyo KEJ, Liu ZH, Magnusson Y, et al. Impact of protein uptake and
degradation on recombinant protein secretion in yeast. Appl
Microbiol Biot 2014;98:7149–59.
Williams M. Progress in Alzheimer’s disease drug discovery: an
update. Curr Opin Invest Dr 2009;10:23–34.
Winderickx J, Delay C, DeVos A, et al. Protein folding diseases and
neurodegeneration: lessons learned from yeast. BBA-Mol Cell
Res 2008;1783:1381–95.
Yao J, Irwin RW, Zhao LQ, et al. Mitochondrial bioenergetic deficit
precedes Alzheimer’s pathology in female mouse model of
Alzheimer’s disease. P Natl Acad Sci USA 2009;106:14670–5.
Zhang W, Espinoza D, Hines V, et al. Characterization of betaamyloid peptide precursor processing by the yeast Yap3 and
Mkc7 proteases. BBA-Mol Cell Res 1997;1359:110–22.