Nervous yeast: modeling neurotoxic cell death

Braun et al. Nervous Yeast: Modeling neurotoxic cell death

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Nervous yeast: 1

Modeling neurotoxic cell death 2

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Ralf J. Braun1, Sabrina Büttner1, Julia Ring1, Guido Kroemer2,3,4, and Frank 4

Madeo1,# 5

1Institute of Molecular Biosciences, Department of Microbiology, Karl-Franzens-University 6

of Graz, Graz, Austria 7 2INSERM, U848; 94805 Villejuif, France 8

3Institut Gustave Roussy, 94805 Villejuif, France 9

4University Paris Sud, Paris-11, 94805 Villejuif, France 10

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Key words: Neurotoxicity, yeast, apoptosis, necrosis, proteinopathy 15

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20 #Corresponding author: 21

Prof. Frank Madeo 22

Institute of Molecular Biosciences 23

Department of Microbiology 24

Humboldtstrasse 50/EG 25

8010 Graz 26

Austria 27

Phone: ++43-316-380-8878 28

Fax: ++43-316-380-9898 29

[email protected] 30

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Abstract 1

Neurodegeneration is characterized by the disease-specific loss of neuronal activity, 2

culminating in the irreversible destruction of neurons. Neuronal cell death can proceed via 3

distinct subroutines such as apoptosis and necrosis, but the underlying molecular mechanisms 4

remain poorly understood. Saccharomyces cerevisiae is an established model for programmed 5

cell death, characterized by distinct cell death pathways conserved from yeast to mammals. 6

Recently, yeast models for several major classes of neurodegeneration, namely α-7

synucleinopathies, polyglutamine disorders, β-amyloid diseases, tauopathies, and TDP-43 8

proteinopathies, have been established. Heterologous expression of the human proteins 9

implicated in these disorders has unraveled important insights in their detrimental function, 10

pointing to ways in which yeast might advance the mechanistic dissection of cell death 11

pathways relevant for human neurodegeneration. 12

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Yeast: a versatile model for cell death and neurodegeneration 1

Neurodegenerative disorders are caused by the specific loss of neuronal activity, 2

which often involves the definitive demise of neurons. Cell death proceeds via different 3

subroutines that are classified according to their etiology or their morphology in accidental 4

cell death (“classical” necrosis), programmed necrosis, apoptosis, or cell death with aberrant 5

autophagy (autophagic cell death) [1]. Each of these types of cell death can contribute to 6

neurodegenerative disorders, following ever more complex molecular pathways. Thus, cell 7

death that manifests with a similar appearance might proceed through biochemically distinct 8

pathways (e.g., caspase-dependent vs. caspase-independent apoptosis) [2]. Reliable in vitro 9

models therefore are required to shed light on the molecular mechanisms of 10

neurodegeneration. 11

Most neurodegenerative disorders are characterized by protein aggregates that 12

accumulate intra- or extracellularly and are thought to critically contribute to cell death [2]. 13

Neurodegenerative diseases can be classified according to the major protein components of 14

their aggregates (Table 1): (i) cytoplasmic α-synuclein-containing Lewy bodies are typical for 15

α-synucleinopathies, including most types of Parkinsonism (PD) [3]; (ii) intracellular 16

aggregation of a disease-specific protein with aberrantly lengthened polyglutamine stretches 17

characterize polyglutamine disorders (e.g., huntingtin in Huntington’s disease; HD [4]) (iii) β-18

amyloid disorders are characterized by the location of the hydrophobic peptide β-amyloid in 19

extracellular plaques (e.g., in Alzheimer’s disease, AD) or in intracellular inclusions (e.g., in 20

inclusion body myositis) [5]; (iv) intracellular inclusions of microtubule-associated protein 21

tau (MAPT; also called tau) characterize tauopathies (e.g., as neurofibrillary tangles in AD) 22

[6]; and (v) TDP-43 proteinopathies are defined by intracellular aggregation of TDP-43 (TAR 23

DNA binding protein) which occurs in some cases of frontotemporal lobar degeneration 24

(FTLD) and the motor neuron disease amyotrophic lateral sclerosis (ALS) [7]. 25


4

During recent years, yeast models have been developed to study all of these 1

neurodegenerative disorders (Figure 1). This review summarizes how yeast can help to 2

decipher molecular mechanisms of neurotoxic cell death. We describe cell death subroutines, 3

pathways and convenient methods for studying cell death in yeast expressing human 4

neurotoxic proteins. Advantages and limitations applying neurotoxic yeast are discussed, and 5

the current state of research for yeast models of the various classes of neurodegenerative 6

disorders is summarized. 7

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Conserved cell death subroutines and pathways in yeast 9

Similar to observations from neurodegeneration research, dying yeast exhibit 10

morphological markers of apoptotic and necrotic cell death [8-10]. In yeast, expression of 11

toxic proteins or exogenous treatment with chemicals, such as hydrogen peroxide and acetic 12

acid, can trigger mitochondrial damage, resulting in the production of reactive oxygen species 13

(ROS) [11]. Oxidative stress and mitochondrial dysfunction are tightly linked (and mostly 14

causative) to diverse yeast cell death scenarios [10, 11], but are also hallmarks of most 15

neurodegenerative disorders, including PD and AD [2]. Similarly, endoplasmic reticulum 16

(ER) stress-mediated death was observed in yeast [12]. For instance, upon persistent 17

accumulation of misfolded proteins in the ER, ROS arise from the ER-resident oxidative 18

protein folding machinery, ultimately resulting in apoptotic cell death [12]. Notably, ER stress 19

is pivotal for the progression of neurodegeneration, such as in HD and ALS [13, 14]. Very 20

recent data demonstrate that autophagy, induced by exogenous application of the polyamine 21

spermidine, relieves oxidative stress and can prevent programmed necrosis in yeast [8]. This 22

is of particular interest as autophagy is implicated in the detoxification of neurotoxic proteins 23

in mammalian cells, for example during the clearance of aggregated superoxide dismutase 1 24

(SOD1) in ALS [14, 15]. 25


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Despite the evolutionary distance between yeast and humans, the underlying 1

molecular players of yeast cell death are surprisingly conserved [11, 16]. Yeast express 2

regulators of cell death, including a yeast caspase (Yca1p), apoptosis-inducing factor (Aif1p), 3

Ndi1p (homolog of ‘AIF-homologous mitochondrion-associated inducer of death’, AMID), 4

Nuc1p (homolog of endonuclease G, EndoG), Nma111p (homolog of HtrA2/Omi), Bir1p 5

(homolog of ‘inhibitor of apoptosis’ IAP family), Bax inhibitor (BI-1), and Cpr3p (homolog 6

of cyclophilin D) [11, 16]. Consistent with findings in mammalian cells, yeast can undgergo 7

distinct caspase-dependent and –independent programmed cell death pathways [16]. For 8

instance, release of Aif1p and Nuc1p, respectively, from permeabilized mitochondria result, 9

like in mammalian cells, in their nuclear translocation causing nuclear DNA fragmentation in 10

a Yca1p-independent fashion [17, 18]. 11

Despite this conservation, the diversity and number of players is reduced in yeast 12

compared to neuronal cells. For instance, yeast expresses only one “classical” caspase 13

(Yca1p) and one IAP family member (Bir1p), whereas neuronal cells express multiple 14

caspases and IAPs [19, 20]. Likewise, conserved cell death pathways are similar, but not 15

identical, between yeast and humans. For example, Yca1p activation depends on ROS, 16

whereas caspase 9 activation depends on the release of cytochrome c into the cytosol [19]. 17

Cytochrome c release has been observed in yeast; however, it remains unclear whether this 18

plays a causative role during cell death or is only a consequence of mitochondrial membrane 19

rupture [11]. Thus, cell death pathways elucidated upon expression of neurotoxic proteins in 20

yeast should be validated in higher model systems, ideally in mammalian neuronal cells. 21

22

Methodological approaches to studying neurotoxic cell death in yeast 23

The effect of neurotoxic proteins on yeast is tested by measuring a reduction in growth 24

or a loss of clonogenic survival, and yeast cell death is resolved with a panel of assays that 25

discriminate various types of cell death (Box 1). Age-related effects on neurotoxicity can be 26


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analyzed (Box 2), and the putative mitochondrial contribution to cell death can be determined 1

by comparing neurotoxicity under obligatory respiratory vs. fermentative growth conditions 2

or in the presence vs. absence of mitochondrial DNA (ρ+ vs. ρ0 strains), as successfully 3

implemented in yeast expressing α-synuclein [21]. 4

Genetic modulators of neurotoxicity can be identified by targeted screens which utilize 5

known yeast cell death genes [21] or by unbiased genome-wide screens [22-24]. Neurotoxic 6

proteins are either expressed in yeast strains which lack known cell death regulators, as was 7

done for α-synuclein [21] or in a collection of yeast knock-out strains that includes all non-8

essential genes [25], successfully performed with α-synuclein and huntingtin [26, 27] (Box 1). 9

When a knockout strain exhibits improved growth and survival upon expression of a 10

neurotoxic protein, a putative pro-death function is attributed to the deleted gene. 11

Alternatively, cDNAs encoding known yeast cell death proteins or a cDNA collection 12

comprising most known yeast open reading frames [28] can be used for co-expressing yeast 13

proteins with neurotoxic peptides, such as α-synuclein [29, 30]. Decreased toxicity and cell 14

death points to a cytoprotective role of the respective yeast protein. 15

In addition, yeast cultures stressed by neurotoxic proteins can be analyzed by 16

transcriptomic and proteomic analyses to identify candidate mRNAs or proteins that modulate 17

or execute toxicity and cell death [31, 32]. Genetic screens and expression profiles are highly 18

complementary approaches and can identify different aspects of toxicity, as recently 19

exemplified for α-synuclein toxicity [32]. Genetic screens tend to identify regulators, whereas 20

mRNA profiling frequently detects metabolic responses. New mathematic algorithms enables 21

bridging of these two approaches, as well as a way to link these techniques to the extensive 22

knowledge of yeast protein–protein interaction networks [32]. 23

24

Yeast proteinopathy models 25


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In recent years, yeast models for multiple neurodegenerative disorders have been 1

established. Although yeast cells, as unicellular organisms, lack many structural and 2

functional hallmarks of neuronal cells that are critical for the progression of 3

neurodegeneration (Box 3), these models have been highly useful in the elucidation of basic 4

cellular mechanisms of toxicity and cell death triggered by human neurotoxic proteins. In this 5

section, we first describe yeast models for α-synucleinopathies and polyglutamine disorders, 6

because these models have been defined most thoroughly and are partially validated in higher 7

model organisms. Although less has been learned from the new yeast models for β-amyloid 8

diseases, tauopathies, and TDP-43 proteinopathies, they offer the potential for providing new 9

insights in neurotoxic mechanisms in the respective human disorders. 10

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α-synucleinopathies 12

α-synuclein and neurodegeneration 13

α-synuclein is a small, abundant protein highly expressed in the central nervous 14

system. Mainly a cytosolic protein, it can bind membranes and is involved in vesicle 15

trafficking; its precise function, however, remains incompletely understood [3]. α-synuclein 16

lacks a defined three-dimensional structure [3]; ranging from unfolded and partially folded 17

conformations, diverse monomeric and oligomeric states to amorphous aggregates, and 18

amyloid-like fibrils, α-synuclein can adapt, structurally, to its surroundings [3]. Aggregated α-19

synuclein is the major constituent of insoluble filaments that form large cytoplasmic 20

inclusions called Lewy bodies [3], a pathological hallmark of the neurodegenerative disorders 21

that are referred to as α-synucleinopathies (e.g., PD, PD with dementia, dementia with Lewy 22

bodies, and multiple system atrophy, Table 1). Missense mutations in SNCA resulting in the 23

expression of α-synuclein variants (A30P, A53T, E46K), as well as increased levels of α-24

synuclein due to gene duplication or triplication can cause PD [3]. Whereas the A53T variant 25

does not disrupt the structure of the folded protein, the replacement of an alanine by the α-26


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helix breaker proline (A30P) interferes with the local helical structure, resulting in a 1

decreased membrane binding [3]. Both variants accelerate α-synuclein aggregation, 2

suggesting common pathogenic mechanisms [3]. 3

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Yeast models for α-synucleinopathies 5

Yeast has been used to dissect normal α-synuclein functions as well as the molecular 6

pathways underlying the cytotoxic consequences of α-synuclein malfunction (Figure 1B). In 7

accordance with other models, α-synuclein is delivered via the secretory pathway and 8

selectively localizes to the plasma membrane [33, 34]. Increased α-synuclein expression 9

induces nucleation-dependent aggregation that is initiated at the plasma membrane, ultimately 10

leading to the formation of cytoplasmic inclusions [33, 34]. Treatment with ferrous ions to 11

induce oxidative stress or with dimethyl sulfoxide (DMSO), which leads to increased 12

phospholipid levels and membrane formation in yeast, strongly enhances α-synuclein 13

aggregation [34]. α-synuclein aggregates cause growth arrest and eventually induce yeast cell 14

death in an expression level-dependent manner [21, 30, 33-35]. 15

Reminiscent of data obtained in higher systems, α-synuclein toxicity in yeast is linked 16

to lipid metabolism and vesicular trafficking [27, 29, 30, 33, 36, 37]. Of note, α-synuclein 17

causes the accumulation of discrete lipid droplets in yeast; the consequences of this effect 18

remain incompletely understood. In addition, α-synuclein was proposed to selectively inhibit 19

phospholipase D in yeast and mammalian cells [33, 38]. However, a recent study suggested 20

that these effects might be caused by increased ER stress following α-synuclein expression, 21

and not through a specific interaction between phospholipase D and α-synuclein [39]. Defects 22

in various vacuolar tethering and sorting complexes cause mislocalization and enhanced 23

inclusion formation of α-synuclein [37]. Consistently, a genome-wide overexpression screen 24

identified proteins involved in vesicle-mediated membrane trafficking as the most efficient 25

suppressors of α-synuclein toxicity [29]. α-synuclein misfolding inhibits ER-to-Golgi 26


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vesicular trafficking and causes a marked increase in ER stress. The overexpression of 1

proteins which facilitate anterograde ER-Golgi trafficking (allowing cells to relieve the α-2

synuclein-promoted transport block) suppressed ER stress and α-synuclein toxicity, whereas 3

the overexpression of proteins that inhibit ER-Golgi trafficking (and hence exacerbate the α-4

synuclein-induced transport block) enhanced α-synuclein toxicity [29]. Overexpression of 5

Ypt1p, a Rab guanosine triphosphatase which functions in ER-Golgi trafficking and 6

associates with α-synuclein inclusions, protected yeast cells from α-synuclein toxicity [29]. 7

Likewise, overexpression of the homologous Rab1 protein protected Caenorhabditis elegans 8

and Drosophila melanogaster from α-synuclein-induced neurodegeneration [29, 40]. 9

Proteasomal dysfunction and chaperone activity have also been implicated in 10

mediating α-synuclein toxicity. Misfolded α-synuclein inhibits the proteasome, and mutations 11

in 20S proteasomal subunits sensitize yeast cells to α-synuclein toxicity [33, 41], whereas, a 12

brief heat shock or the enhanced expression of the Hsp70 chaperone Ssa3p protects against α-13

synuclein toxicity [35]. 14

Data obtained in cell culture and animal models suggest that α-synuclein toxicity 15

culminates in oxidative stress, mitochondrial dysfunction and cell death [42]. Consistently, 16

expression of α-synuclein in yeast results in apoptotic as well as in necrotic death [21, 35]. 17

Massive ROS accumulation, cytochrome c release, phosphatidylserine externalization, DNA 18

fragmentation, and necrotic loss of membrane integrity are all associated with α-synuclein 19

expression [21, 35]. The toxicity of α-synuclein in chronologically aged yeast is independent 20

of the apoptotic proteins Yca1p, Aif1p and Nma111p [21]. Moreover, although ER stress is a 21

consequence of α-synuclein toxicity, α-synuclein triggered cell death is also independent of 22

the ER stress regulators Ire1p and Hac1p [21]. By contrast, loss of mitochondrial DNA (and 23

therefore of oxidative phosphorylation) delays α-synuclein-induced ROS production and 24

death, pointing to an essential role for mitochondria in the cytopathic effect of α-synuclein 25

[21]. Whereas death during chronological aging is YCA1-independent [21], Yca1p is involved 26


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in death of α-synuclein-expressing during exponential growth [35], therefore implicating age-1

related differences in the cell death mechanism. 2

The α-synuclein A30P and A53T variants have been studied in yeast. A53T resembles 3

wild-type α-synuclein with respect to membrane interaction, localization, and aggregation, 4

mainly provoking the same (or slightly enhanced) effects on lipid metabolism, vesicular 5

trafficking, proteasomal dysfunction, toxicity, and eventually cell death [21, 33]. By contrast, 6

A30P fails to enter the secretory pathway and therefore is not targeted to the plasma 7

membrane, but instead displays a diffuse localization throughout the cytosol without forming 8

cytoplasmic inclusions [33, 34]. Thus, impaired membrane binding of A30P seems to prevent 9

its aggregation [33, 34, 43]. Although A30P blocks vesicular trafficking to the same extent as 10

wild-type α-synuclein and A53T, it does not trigger the accumulation of lipid droplets [33, 11

34]. The toxic effects of A30P in yeast remain controversial. Whereas some results indicate 12

that A30P is as cytopathic as wild-type α-synuclein and A53T [30, 35, 41, 44], other studies 13

report limited or no toxicity for A30P [33, 43, 45]. These variations might be caused by 14

different expression levels, as enhanced α-synuclein expression correlates with enhanced 15

toxicity [33, 45]. 16

Although yeast does not have an α-synuclein homologue, orthologues to other 17

Parkinson genes exist. Recently, a genetic interaction between α-synuclein and YPK9, the 18

yeast orthologue to human PARK9 (ATP13A2), was established in yeast and validated in 19

animal models of PD [46]. YPK9 and PARK9 (ATP13A2) protect cells from α-synuclein 20

misfolding and manganese toxicity, a known environmental risk factor for PD [46]. 21

Altogether, α-synuclein toxicity in yeast is characterized by ER stress, oxidative stress, 22

and impaired protein degradation, culminating in mitochondrion-dependent apoptotic and 23

necrotic cell death. Moreover, yeast is a potent tool for elucidating the interconnections 24

among α-synuclein-triggered cell death, protein folding and aggregation, membrane-binding, 25

and functional interactions with other Parkinson-related genetic and environmental factors. 26


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Polyglutamine disorders 2

Polyglutamine proteins and polyglutamine disorders 3

Some human genes contain CAG codon repeats which encode polyglutamine tracts. In 4

nine such genes, abnormally increased numbers of these repeats result in the expression of 5

aggregation-prone proteins with expanded polyglutamine stretches that cause the so-called 6

polyglutamine disorders (Table 1). These include HD, a movement disorder characterized by 7

intranuclear and cytoplasmic inclusions of the polyglutamine protein huntingtin and six 8

different types of spinocerebellar ataxias [4]. 9

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Yeast models for polyglutamine disorders 11

In yeast, expression of huntingtin exon I fragments comprising the polyglutamine 12

stretches faithfully recapitulates huntingtin aggregation in a polyglutamine length-dependent 13

manner [33, 47-49]. Huntingtin aggregation does not correlate with cellular toxicity in all 14

cases, but instead strongly depends on both sequences flanking the polyglutamine stretches 15

(i.e., cis-acting elements), as well as the presence of a specific protein interaction network 16

within different yeast strains (i.e., trans-acting elements) [50, 51]. Although it does not 17

prevent the formation of aggregates, the proline-rich domain adjacent to the polyglutamine 18

domain prevents overt cytotoxicity of the huntingtin fragment [50-52]. Indeed, this domain 19

serves as a target sequence for the efficient formation of large perinuclear aggregates, called 20

aggresomes [53], which are actively formed at the centrosome near the nucleus and might be 21

part of a cytoprotective mechanism that is activated upon overload of the protein folding and 22

degradation pathways [54]. Accordingly, yeast cells which contain aggresome-like 23

polyglutamine aggregates demonstrate higher viability than cells with multiple, smaller 24

aggregates [51, 53] (Figure 1C). 25


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Cdc48p, which plays a critical role in the ubiquitin-dependent ER-associated protein 1

degradation pathway (ERAD), is essential for the formation of cytoprotective polyglutamine 2

aggresomes in yeast [53]. By contrast, cytotoxic polyglutamine aggregates might entrap 3

functional Cdc48p leading to the decreased formation of polyglutamine aggresomes and to 4

ER stress resulting from impaired ERAD [55]. Indeed, the human orthologue of Cdc48p, 5

valosin-containing protein (VCP), interacts with aberrant polyglutamine proteins, modulating 6

their aggregation and toxicity in mammalian cells, as well as in Drosophila and C. elegans 7

[56-58]. However, it remains elusive how the underlying cellular mechanism in yeast 8

corresponds to the results obtained in these models. When expressed in yeast, UBB+1, a 9

frameshift variant of human ubiquitin B that accumulates in polyglutamine disorders [59], 10

prevents efficient aggresome formation of disease-associated huntingtin [60]. Thus, the 11

Cdc48p- and UBB+1-mediated switch between cytotoxic huntingtin aggregates and 12

cytoprotective aggresomes that is observed in yeast might be relevant for HD. 13

In yeast, aggregated huntingtin co-localizes with autophagic markers at perivacuolar 14

sites, suggesting a role for autophagy in the clearance of polyglutamine protein aggregates 15

[61]. Such a neuroprotective function of autophagy has been confirmed in a Drosophila 16

model of HD [62]. Intriguingly, a recent study demonstrated that increased autophagy relieves 17

programmed necrosis, and prolongs longevity, in yeast, flies and worms [8]. 18

Yeast prions play an important role in the formation and propagation of disease-19

associated huntingtin aggregates [47, 48, 63-65]. For instance, [PIN+], the prion form of 20

Rnq1p, is necessary for both aggregation and toxicity of aberrant polyglutamine proteins [48, 21

63]. Both prion and polyglutamine aggregate formation are modulated by chaperones [64]; for 22

example, the disaggregase Hsp104p enables polyglutamine aggregate expansion and yeast 23

prion propagation [47, 52]. Chaperones therefore affect polyglutamine toxicity either directly 24

via modulation of polyglutamine aggregates, or indirectly through modulation of prions that 25

functionally interact with polyglutamine aggregation [64]. 26


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Most studies that address sequence-specific effects of huntingtin constructs or 1

aggregation- and toxicity-modulating effects of yeast prions and chaperones, have limited 2

themselves to growth measurement; only a few studies have determined the capacity of 3

polyglutamine proteins to kill yeast cells [55, 66-69]. Solans and colleagues [66] observed 4

that disease-associated huntingtin caused a progressive reduction in the function and 5

abundance of mitochondrial respiratory chain complexes II and III, leading to reduced 6

oxidative phosphorylation and enhanced ROS production. This effect might be explained by 7

the detrimental interaction of polyglutamine aggregates with mitochondrial networks [66]. 8

These data are in line with results from mammalian cell culture and animal models for HD 9

that suggest a crucial mitochondrial contribution in polyglutamine-triggered cell death [70]. In 10

these studies, huntingtin interacts with mitochondria, impairs respiration, mitochondrial 11

membrane potential, and mitochondrial dynamics, resulting in bioenergetic failure, oxidative 12

stress and ultimately, cell death [70]. 13

Oxidative stress can be tightly connected to ER stress in both yeast and mammalian 14

cells [12]. Intriguingly, yeast and neuronal cells expressing disease-associated huntingtin 15

demonstrate a specific and marked defect in ERAD, leading to the induction of the unfolded 16

protein response and ER stress. In these studies, impaired ER, but not cytosolic, protein 17

homeostasis was critical for polyglutamine-triggered cell death; moreover, ERAD deficiency 18

and the subsequent ER stress could be attributed, at least in part, to the entrapment of the 19

pivotal ERAD component Cdc48p (VCP) [55]. Indeed, Cdc48p depletion results in ER stress 20

and mitochondrion-dependent apoptotic cell death in yeast [10, 31, 71, 72]. Therefore, 21

Cdc48p (VCP) might constitute the switch for both mitochondrion- and ER-dependent cell 22

death in yeast and neuronal cells expressing pathogenic polyglutamine proteins. 23

In summary, yeast models for polyglutamine disorders allowed the elucidation of 24

aggregation mechanisms of polyglutamine proteins. Upon polyglutamine toxicity, several 25

pathways, including ER stress, oxidative stress, and mitochondrial dysfunction contribute to 26


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(apoptotic) cell death in yeast. It will be important to further evaluate how critical regulators 1

of apoptotic, necrotic, or autophagy-related cell death might contribute to the cytopathic 2

effects of polyglutamine proteins in yeast. 3

4

β-amyloid disorders 5

β-amyloid and neurodegeneration 6

β-amyloid is a peptide that is generated by sequential proteolysis of the amyloid 7

precursor protein (APP) by β- and γ-secretases [5]. The major species of β-amyloid are the 40 8

amino acid long Aβ40 peptide and the more hydrophobic Aβ42 peptide. Mutations in APP or 9

components of the γ-secretase complex result in increased Aβ42 production [5] which leads to 10

β-amyloid oligomerization and the formation of stable amyloid-like deposits that are 11

characteristic of β-amyloid disorders, including AD, Down syndrome, cerebral amyloid 12

angiopathy, and inclusion body myositis [5] (Table 1). In AD, β-amyloid deposits are 13

predominantly found in the extracellular space of the brain and are called plaques. 14

Intracellular β-amyloid is found in the secretory pathway, in autophagosomes, in the cytosol, 15

and at mitochondria [73], thus potentially affecting diverse cellular processes. 16

17

Yeast models for β-amyloid disorders 18

Yeast has been used for the evaluation of the cellular consequences of both extra- and 19

intracellular β-amyloid (Figure 1D). Extracellular oligomeric (but not fibrillar) β-amyloid 20

kills yeast in a dose-dependent fashion, as evidenced by decreased clonogenicity of treated 21

cultures; however, the molecular mechanisms of amyloid uptake and its subsequent toxicity 22

remain elusive [74]. 23

The accumulation of intracellular β-amyloid has been suggested to contribute to 24

cellular toxicity during AD progression [73]. Therefore, several groups established yeast 25

models which express high levels of intracellular β-amyloid [75-78]. Although the turnover of 26


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β-amyloid peptides in yeast is rather high, the β-amyloid peptide can be stabilized by the 1

fusion to large protein tags [76-78]. Cain et al. expressed an Aβ42–GFP fusion protein that 2

localizes to the yeast cytosol, and in contrast to unmodified GFP, this fusion protein is 3

predominantly found in the insoluble protein fraction, hinting at its Aβ42-dependent 4

aggregation [76]. Accordingly, immunoelectron microscopy identified Aβ42-GFP within 5

amorphous patches [76]. Aβ42-GFP aggregation could be decreased upon treatment with 6

folinic acid, a synthetic form of folate; however, the underlying molecular mechanisms for 7

this process remain unknown [75]. Notably, dietary folate deficiency constitutes a risk factor 8

for AD. 9

In contrast to extracellular oligomeric β-amyloid, yeast cells tolerate intracellular APP 10

fragments including β-amyloid peptides; indeed, Aβ42 fusion proteins are non-toxic to yeast 11

[75-78]. Although Aβ42-GFP expression causes a small decrease in growth rate and induces 12

the heat shock response [76], it fails to induce morphologic markers of cell death or to reduce 13

clonogenicity. Notably, studies from other model systems suggest that β-amyloid triggers 14

ROS generation and impairs mitochondrial, proteasomal, and autophagic activities. Therefore, 15

future studies are needed to address how yeast cells are able to tolerate A42 expression; this 16

could be accomplished by screening a yeast gene deletion library for genes that are important 17

for the tolerance and detoxification of Aβ42. 18

19

Tauopathies 20

Tau and neurodegeneration 21

Tau (MAPT) is a neuronal microtubule-associated protein that promotes microtubule 22

assembly and stabilization and contributes to axonal maintenance and transport [5, 6]. 23

Alternative splicing results in proteins which contain either three (tau 3R) or four (tau 4R) 24

microtubule-binding domains; tau 4R demonstrates higher affinity to microtubules than 25

tau 3R. Tau–microtubule binding is negatively regulated via phosphorylation. Whereas the 26


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proline-directed kinase GSK3β (glycogen synthase kinase 3β) phosphorylates tau, the 1

activation of Cdk5 (cyclin-dependent kinase 5) results in decreased tau phosphorylation [6]. 2

In AD and a subtype of FTLD which are referred to as ‘tauopathies’ (Table 1), tau 3

misfolds into filaments that can develop into intracellular neurofibrillary tangles [5, 6]. In 4

FTLD, disease-associated variants of tau, including the most frequent variant P301L, can 5

affect the microtubule binding domain, resulting in decreased tau–mircrotubule binding and 6

increased tau aggregation [5, 6]. In tauopathies tau also is hyperphosphorylated and 7

extensively truncated. In vitro studies suggest that this hyperphosphorylated and truncated 8

protein assembles into tau filaments more efficiently than the native protein. Tau cleavage is 9

mediated by known cell death proteases, namely caspases, the calcium-activated protease 10

calpain, and the lysosomal protease cathepsin D [6], suggesting that apoptosis, necrosis and 11

autophagy, respectively, might be connected to the pathogenesis of tauopathies. However, the 12

significance of the different types of cell death during neuronal loss in tauopathies remains 13

highly controversial, as contradictory results have been obtained from multiple tauopathy 14

models including cell culture and animal models [6]. These discrepancies highlight the need 15

for a better understanding of the interrelation between tau modification, aggregation, and 16

eventually, cell death. 17

18

Yeast models for tauopathies 19

Yeast does not possess an orthologue to human tau, but human tau 4R and tau 3R can 20

be expressed in yeast [79]. Phosphorylation of ectopically expressed tau is regulated by the 21

yeast kinases Mds1p and Pho85p, the orthologues of human GSK3β and Cdk5, respectively, 22

and occurs at the same residues that are hyperphosphorylated in neurons. Consistent with 23

results obtained in the mammalian system, hyperphosphorylation correlates with the 24

formation of insoluble tau aggregates [79] (Figure 1E). Although human tau does not 25

appreciably bind yeast microtubules, efficient binding of purified human tau, heterologously 26


17

expressed in yeast, to mammalian microtubules is observed in vitro. Consistent with data 1

from mammalian cells, this binding is inversely regulated by the tau phosphorylation state 2

[80]. Nevertheless, the lack of tau-binding to yeast microtubules excludes the use of this yeast 3

model to analyze deleterious effects of aberrant tau on the microtubule cytoskeleton in vivo 4

and its consequences on cell survival. Yet, this model can be used to elucidate toxic gain-of-5

function effects of aberrant tau which potentially trigger cell stress and cell death independent 6

of its normal function in regulating the microtubule cytoskeleton. 7

In fact, both wild-type and FTLD-associated tau 4R-P301L fail to affect the viability 8

of normal yeast cells, but tau 4R-P301L reduces the growth of Δpho85 cells, in which it is 9

hyperphosphorylated and aggregated. Whether the growth delay is due to a consequent 10

induction of cell death remains unknown [34]. Interestingly, synergistic toxicity is observed, 11

both in mammals and in yeast, upon co-expression of tau and α-synuclein [34, 81]. Thus, 12

yeast might facilitate the analysis how toxic hyperphosphorylated tau interacts with other 13

neurotoxic proteins or potential disease-modulating factors, such as UBB+1, a detrimental 14

ubiquitin variant that accumulates in tauopathies [59]. 15

16

TDP-43 proteinopathies 17

TDP-43 and neurodegeneration 18

TDP-43 is involved in pre-mRNA splicing, mRNA processing and transport, and 19

transcription. It is the major component of ubiquitin-positive intracellular inclusions in FTLD-20

U, a subtype of FTLD, and in many cases of ALS [7] (Table 1). FTLD is the second most 21

frequent form of presenile dementia after AD, whereas ALS constitutes the most common 22

motor neuron disease [7]. FTLD, ALS and other neurodegenerative disorders with TDP-43 23

inclusions (e.g., AD, Table 1) have been referred to as TDP-43 proteinopathies [7]. In 24

diseased brains, TDP-43 forms non-amyloid intracellular inclusions that are enriched with 25

characteristic hyperphosphorylated and ubiquitylated C-terminal fragments of TDP-43 [7]. 26


18

Interestingly, the C-terminal part of the protein is mostly affected by mutations in TDP43 that 1

have been identified in patients with ALS [7]. Accordingly, expression of TDP-43 fragments 2

in a mammalian cell culture model results in the formation of ubiquitin-positive cellular 3

inclusions correlated with increased incidence of cell death [82]. However, whether TDP-43 4

fragmentation and modification is the cause or a consequence of TDP-43 aggregation and 5

how this relates to its toxicity remains elusive [7]. 6

7

Yeast model of TDP-43 proteinophathy 8

Similar to what is observed in human TDP-43 proteinopathies, yeast expressing 9

human TDP-43 exhibit cytoplasmic TDP-43 aggregates [83] (Figure 1F). TDP-43 aggregation 10

correlates with increased signs of toxicity [83], namely (i) a marked decrease in growth in 11

spotting assays, (ii) decreased clonogenic potential, and (iii) an increased number of dead 12

cells that stain with the vital exclusion dye propidium iodide. However, it remains unknown 13

whether cell death proceeds via apoptosis or necrosis and which molecular players are 14

involved. 15

Deletion mapping revealed that most of the C-terminal region of TDP-43 is required 16

for its aggregation and toxicity [83]. Intriguingly, most ALS-associated TDP-43 variants are 17

characterized by single amino acid substitutions in this portion of the protein [7]. In yeast, the 18

expression of ALS-associated TDP-43 variants results in increased incidences of multiple 19

aggregate formation as compared to expression of wild-type TDP-43 [84]. Most importantly, 20

this observation correlates with augmented toxicity, as the growth deficit of cultures 21

expressing ALS-associated TDP-43, compared to wild-type TDP-43, is increased [84]. As C-22

terminal TDP-43 fragments and disease-associated TDP-43 variants are hallmarks of TDP-43 23

pathology in diseased brains [7], these findings underline the probable utility of the TDP-43 24

yeast model. 25


19

Notably, TDP-43 aggregates are biochemically and biophysically distinct from 1

aggregates observed in yeast HD and yeast prion models [83, 84]. As the known modulators 2

of toxicity in yeast models for HD and α-synucleinopathies also significantly differ [83], this 3

finding strongly hints to specific cytopathic effects of each individual neurotoxic protein in 4

yeast. Therefore, yeast might contribute to the elucidation of molecular effectors that are 5

specific for these different classes of neurodegenerative disorders. 6

7

Concluding remarks 8

Yeast is an established model organism for the analysis of conserved cell death 9

pathways such as apoptosis and necrosis. The facile measurement of cell viability, cell stress 10

and cell death and the high genetic tractability of this organism enable a time- and cost-11

effective exploration of cell death pathways. Several hallmarks of neurodegenerative 12

disorders, namely protein aggregation, increased cell death, oxidative stress, mitochondrial 13

damage, ER stress and impaired proteasomal and autophagic protein degradation can be 14

advantageously analyzed in yeast in a high-throughput manner. Multiple yeast models have 15

been generated for the mechanistic exploration of major human neurodegenerative disorders, 16

namely α-synucleinopathies, polyglutamine disorders, β-amyloid disorders, tauopathies, and 17

TDP-43 proteinopathies. To date, these models have been successfully employed for the 18

elucidation of (i) normal cellular functions of neurotoxic proteins; (ii) cellular mechanisms of 19

protein aggregation; and (iii) their toxicity which triggers stress and cell death. Notably, 20

results obtained in these yeast models demonstrate a high degree of specificity for different 21

neurotoxic proteins and have, in part, been validated in higher model organisms. Therefore, it 22

seems plausible that yeast models of neurodegenerative proteinopathies, for instance through 23

applying a systematic analysis of the involvement of known yeast cell death genes, could lead 24

to the discovery of additional disease-relevant genes and processes. We anticipate that such 25

genes could be critically involved in disease-specific molecular cell death pathways thereby 26


20

enabling targeted approaches to inhibit neuronal cell loss in human neurodegenerative 1

disorders. 2

3


21

Acknowledgments 1

We are grateful to the German Research Foundation (DFG) for grant BR 3706/1-1 for R.J.B. 2

(DFG post-doc fellowship), to the Austrian Science Fund (FWF) for grant T-414-B09 to S.B 3

(Hertha-Firnberg fellowship), for grant S-9304-B05 to F.M., J.R. and S.B., and for grant 4

“Lipotox” to F.M. and S.B., and to the European Commission for project APOSYS to F.M. 5

and G.K. 6

7


22

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48 49

50


26

Figure legends 1

Figure 1: 2

Yeast cell death models for human neurodegenerative disorders. 3

The different panels describe pivotal molecular players that are involved in the aggregation 4

and modification of neurotoxic protein aggregates, and in the progression of cell death. 5

Cellular stresses are highlighted with yellow flashes, and organelles involved in neurotoxicity 6

are accentuated with bright colors. 7

(A) Yeast models for cell death research. Expression of neurotoxic proteins can result in 8

cellular stresses, including the heat shock response, mitochondrial and ER stress, as well as 9

oxidative stress (via ROS) and vacuolar/autophagic dysfunction. Such stress can trigger 10

different pathways of cell death that involve Yca1p, Aif1p, and cytochrome c. 11

(B) Yeast models for α-synucleinopathies. α-synuclein binds the plasma membrane, triggering 12

cytoplasmic oligomerization and fibrillization. Misfolded α-synuclein inhibits vesicular 13

trafficking between the ER and Golgi which can be relieved by overexpression of the small 14

GTPase, Ypt1p. Apoptosis is linked to oxidative stress (via ROS), pivotal mitochondrial 15

damage and ER stress due to impaired vesicle trafficking. Yca1p only contributes to cell 16

killing when the cells are in logarithmic growth phase. 17

(C) Yeast models for polyglutamine disorders. Proteins with expanded polyglutamine 18

residues form chaperone- and prion-dependent oligomeric and fibril-like aggregates, causing 19

damage to mitochondria and the ER, leading to oxidative stress and cell death. Oligomeric 20

aggregates can be partially detoxified by transporting them to perinuclear and perivacuolar 21

collection points. The ER quality control protein Cdc48p promotes and the human aberrant 22

ubiquitin variant UBB+1 interfere with the formation of such perinuclear aggresomes. 23

(D) Yeast models for β-amyloid disorders. Extracellular β-amyloid oligomers cause toxicity, 24

through unknown mechanisms. Intracellular β-amyloid is only stable when fused to a large 25

protein tag, such as GFP, triggering a heat shock response. 26


27

(E) Yeast models for tauopathies. Phosphorylation of human tau expressed in yeast is 1

regulated by Pho85p and Mds1p, the yeast homologues of human tau kinases. Tau forms 2

tangle-like filaments in yeast, yet has mild or no effects on cell survival. 3

(F) Yeast models for TDP-43 proteinopathies. Expression of human TDP-43 in yeast results 4

in nuclear and cytoplasmic aggregation, decreased growth and increased death. The molecular 5

mechanisms of stress and death remain elusive. 6

7


28

Tables 1

2 3 Table 1: Classes of proteinopathies and their corresponding disorders 4

Proteinopathy

Major component of protein aggregates

Human disorders

β-amyloid disorder β-amyloid peptides

Alzheimer’s disease, cerebral amyloid angiopathy, Down syndrome, inclusion body myositis

Tauopathies MAPT (also called tau)

Alzheimer’s disease, frontotemporal lobar degeneration: Frontotemporal dementia with Parkinsonism linked to chromosome 17, Pick’s disease, corticobasal degeneration, progressive supranuclear palsy, neurofibrillary tangle dementia, argyrophilic grain disease, sporadic multiple system tauopathy with dementia

α-synucleinopathies

α-synuclein Parkinson’s disease, Parkinson’s disease with dementia, dementia with Lewy bodies, Hallervorden-Spatz syndrome, multiple system atrophy

Polyglutamine disorders

Disease-specific protein with expanded polyglutamine stretches, e.g., huntingtin

Huntington disease, dentatorubropallidoluysian atrophy, spinobulbar muscular atrophy, spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17

TDP-43 proteinopathies

TDP-43 Frontotemporal lobar degeneration with ubiquitin-positive inclusions, amyotrophic lateral sclerosis, Alzheimer’s disease, dementia with Lewy bodies, argyrophilic grain disease, Perry syndrome, Huntington’s disease, inclusion body myositis

5 6

7


29

Box 1: 1

Assaying viability and cell death in yeast. 2

The toxicity of neurotoxic proteins in yeast is tested by growth assays on agar plates or 3

in liquid cultures [22-24]. Yeast strains are transformed with constructs which encode 4

neurotoxic proteins under the control of inducible promoters. Cultures are spotted in serial 5

dilution on agar plates with selective medium, ensuring maintenance of the constructs and 6

repression or induction of protein expression, (Figure Ia). After two days of incubation, a 7

decrease in growth upon protein expression compared to inert vector controls indicates 8

toxicity. Although this is a simple and fast method which can facilitate the use of genome-9

wide screens, such growth assays cannot distinguish between decreased growth rates and 10

increased cell death. Instead, it is preferable to use a clonogenic assay to measure the 11

percentage of dead cells in yeast cultures [17, 19]. A defined number of cells is plated on agar 12

plates with ideal nutrient composition, i.e., rich medium that represses protein expression and 13

does not select for plasmid maintenance (Figure Ib). The number of colonies (colony forming 14

unit, CFU) is determined after two days of incubation and correlates with the survivability of 15

the plated yeast culture. Low CFU numbers, i.e., a decrease in clonogenicity of cultures 16

expressing neurotoxic proteins, indicates increased cell death or cell cycle arrest. 17

To distinguish among different types of cell death and cell cycle arrest, cultures are 18

analyzed with assays measuring morphological markers of cell death (Figure Ib). Apoptotic 19

yeast cells can be visualized by Annexin V staining which detects the externalization of 20

phosphatidylserine to the outer layer of the plasma membrane, or with ‘terminal 21

deoxynucleotidyl transferase dUTP nick end labeling’ (TUNEL) that visualizes DNA 22

fragmentation [9]. The chemical propidium iodide (PI) enters dead yeast cells owing to their 23

ruptured cellular membranes [17]. Yeast cells that only stain for Annexin V are apoptotic, 24

cells that only stain for PI are necrotic, and Annexin V/PI double-positive cells can be 25

discriminated as secondary necrotic or late apoptotic [17]. Autophagic activities that might 26


30

contribute to cell death are determined with an alkaline phosphatase (ALP) reporter assay. 1

This colorimetric assay measures the activity of the yeast alkaline phosphatase (Pho8p-ΔN60) 2

that is expressed in the cytosol and becomes activated upon autophagic translocation to the 3

vacuole [8]. Increased levels of ROS, which correlate with apoptotic and necrotic cell death in 4

yeast, are detected with ROS-sensitive stains, such as dihydroethidium (DHE) [10]. 5


31

Box 1, Figure I: 1

Evaluation of growth, survival and cell death upon expression of neurotoxic proteins. 2

Flow chart illustrating the multiple experimental approaches to analyze neurotoxicity and 3

neurotoxic cell death and cell stress in yeast. 4

5


32

Box 2: 1

Aging of neurotoxic yeast. 2

Aging is the most important risk factor for the development of neurodegenerative 3

disorders, including PD, HD, and AD [2]. Yeast is a valuable model to study processes of 4

replicative and chronological aging [85]. The yeast replicative life span, or mother cell-5

specific aging, is defined as the number of divisions an individual cell undergoes before 6

dying, whereas the yeast chronological life span is the length of time a population remains 7

viable in stationary phase, mimicking the situation of post-mitotic cells, such as neurons [85]. 8

Both replicative and chronological aging of yeast cultures trigger apoptosis and necrosis [85, 9

86]. Therefore, the rapid aging of yeast, coupled with a molecular understanding of both 10

replicative and chronological aging processes, allows the analysis of age-dependent 11

neurotoxic cell death in a fast and convenient manner [85, 86]. For determining the replicative 12

life span of mother cells expressing neurotoxic proteins, daughter cells are repeatedly 13

removed from the aging mother cells, and the number of budding events is counted. During 14

chronological aging of yeast cultures expressing neurotoxic proteins, samples from different 15

time points are analyzed for decreased survival applying a clonogenic approach, and for the 16

emergence of stress and cell death markers (see Box 1). Indeed, chronologically aged yeast 17

has been successfully applied to elucidate mitochondrion-dependent apoptotic and necrotic 18

cell death upon α-synuclein expression [21]. 19

20


33

Box 3: 1

Advantages and limitations using neurotoxic yeast models. 2

Although yeast offers many advantages for mechanistic dissection of neurotoxic cell 3

death (e.g., clonogenic assays), there are some natural limitations: Yeast as unicellular 4

organisms are not embedded in a tissue like neurons, nor are they functionally linked to other 5

cells. Yeast lack neuron-specific morphological structures, such as dendrites, axons and 6

synapses. Consequently, the underlying neuron-specific molecular inventories are missing, 7

including homologues of some human neurotoxic proteins, such as α-synuclein and TDP-43. 8

Neurons are highly specialized, differentiated and post-mitotic, whereas yeast cells adapt 9

easily to altered environmental conditions and divide when sufficient nutrients are provided. 10

As compared to yeast, neurons express a more diverse collection of molecular cell death 11

players. For instance, yeast cells possess only one “classical” caspase (Yca1p), whereas 12

neurons express multiple caspases differentially involved in regulating cell death [16, 19]. A 13

principle complication for all neurotoxic cell death models (including neurotoxic yeast) is the 14

possibility that neuronal cell death might not be pivotal for neurodegeneration, but instead just 15

a consequence of neuronal dysfunction (e.g., synaptic dysfunction) triggered by neurotoxic 16

proteins. 17

The reduced complexity of yeast as compared to neuronal cells is, however, an 18

important advantage of neurotoxic yeast models. The yeast genome is compact, and 19

comprehensive yeast gene deletion and cDNA libraries are available [25, 28]. Although 20

homologues of some neurotoxic proteins are missing in yeast, their molecular interaction 21

networks are surprisingly conserved. For example, α-synuclein functionally interacts with 22

yeast proteins involved in vesicular trafficking, consistent with its proposed function in 23

neuronal vesicular transport [29]. Many cell death pathways in yeast are simplified but 24

principally conserved to neurons. For instance, oxidative stress and the pivotal mitochondrial 25

contribution to cell death can be observed in neurotoxic yeast models, such as those for α-26


34

synucleinopathies and polyglutamine disorders, as well as in neurons [2, 21, 66]. Neurons 1

demonstrate a high energy turnover requiring respiration. Yeast, in contrast to neuronal cell 2

cultures, can efficiently be switched from fermentation to respiration by changing the growth 3

media to an obligatory respiratory carbon source, such as lactate, mimicking the metabolism 4

of neurons in the brain. Accordingly, yeast cells switch from a dividing in a post-mitotic state 5

upon chronological aging, mimicking the neuronal situation (see Box 2). 6

7

P

PP

PP P

P

PPP

P

PPP

P

P

PP

Pho85pPP

Chaperones

ROS

Prion

Cdc48p

Yca1p

Proteasome

ROS

Yca1p

Heat shock response

ER stress

Aif1p

Apoptosis

Monomer

Monomer

P

PP

P

P

PP

P

P

P

PP

P Phosphate(e)

(b)

(c)

Heat shock response

ROS

ER stress

Yca1p

Apoptosis

Apoptotic yeast

β-Amyloid

Tau

α-Synuclein

Polyglutamine

TDP-43

Oligomer

Fibril

Monomer

Oligomer

Fibril

Monomer

Oligomer

Fibril

Monomer

Oligomer

Fibril

GFP

Figure 1

(a)

(d)

(f)

ERNucleus

Mitochondria

Vacuole

Cytochrome c

Golgi

Tangle

Mds1p

Nucleus

Mitochondria

Vacuole

Mitochondria

ER

ER

Nucleus

Nucleus

Proteasome

Golgi

UBB+1ER stress

Ypt1p

Date post:	30-Apr-2023
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Nervous yeast: modeling neurotoxic cell death

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