WO2015066776A2 - A yeast model for synergistic toxicity - Google Patents

Abstract

Disclosed herein are yeast cells which co-express both Αβ and tau and show greater cytotoxicity than cells expressing Αβ or tau alone. These yeast cells model the synergistic effects of Αβ and tau, and are useful for identifying compounds that reduce Αβ and tau toxicity and for obtaining hyperphosphorylated tau species.

Description

A YEAST MODEL FOR SYNERGISTIC TOXICITY

FIELD OF I NVENTION

This invention relates to yeast cells expressing Αβ and tau. The yeast ceiis are suitable for analysis of various aspects of Αβ and tau pathology, such as toxicity mechanisms underlying pathologies, identification of molecules which reduce toxic effects of Αβ and tau, and formation of hyperphosphorylated tau species.

BACKGROUND

Alzheimer's Disease (AD} is a leading ca use of dementia in the elderly population. Both heredita ry and sporadic forms of AD have been identified and studied extensively, but there is no cure for AD. Current medications provide only symptomatic relief, in pa rt because the fundamental mechanisms underlying AD onset and progression remain unclear. AD is characterized by two distinct ha llmarks: extracellular a myloid plaques, consisting mainly of the Αβ₄? peptide (Glenner & Wong, 1984; Masters et al, 1985), and intracellular neurofibrillary tangles (NFTs) consisting of the Ta u protein (Goedert et a 1, 1988; Grund ke-iqbal et al, 1986; Sergeant et al, 2005). The interplay between these two proteins is pivotal in the progression of the disease, but the exact nature of the molecular interactions is not clear. Three majo r hypotheses of how neuronal toxicity and eventua l death arise are commonly cited (Ittner & Gotz, 2011). The first hypothesis follows the classical amyloid cascade theory, where an increased generation of Αβ₄₂, either through mutations or through the effects of an altered cellular metabolism, leads to intracellular amyloid oligomers (Hardy & Selkoe, 2002). These oligomers can disrupt synaptic transmission by binding to N DA receptors (Oddo et al, 2003 ), inhibiting the proteasome (Almeida et a I, 2006), damaging mitochondria (Caspersen et a I, 2005; Keil et al, 2006; Ma ncza k et al, 2006) and by interfering with Ca²* homeostasis (Moussa et al, 2006). The cellular stress this causes will lead to an altered activity of kinases and phosphatases, hyperphosphorylating Tau and eventually causing oSigomerization of Tau and the formation of paired helical fragments and NFTs (Lewis et a I, 2001; Terwel et al, 2008) . I n this hierarchical mode of interaction, Αβ can be considered the trigger, and Tau the effector of neuronal death. A second hypothesis states that Α « a nd Tau can synergisticaily inhibit the same process or organelle by interacting at different sites. Both proteins will, for exa mple, target a different complex in the respiratory chain, thereby causing more disruption than either could do on its own (Rhein et ai, 2009). The result of this mitochondrial dysfunction is a feedback loop where mitochondrial damage in turn causes more amyloid and Tau cytotoxicity. In a third mode of action, Tau acts as the mediator of amyloid toxicity, i.e. toxic effects caused by Αβ,₂ are prevented in the absence of Tau (Rapoport et al, 2002). This occurs because Tau can act as a postsynaptic scaffolding protein, effectively bringing the proteins necessary for amyloid-induced excitotoxicity in close proximity to each other, A dendritic localization of Tau is essential in this scenario, a n event that is thought to happen in later stages of the disease process (Ittner et al, 2010}.

Yeast cells have already played an important role in the elucidation of basic cellular processes like vesicular trafficking and the regulation of the ceil cycle. More recently, yeast has emerged as a powerful tool in the study of neurodegenerative disease. Yeast models have been established for AD, Parkinson's disease and Huntington's disease, among others (Fra nssens et al, 2013; Mager and Winderickx, 2005; Winderickx et a 1, 2008). For AD, the primary focus was to use yeast to study amyloid precursor protein (APP) metabolism (Edbauer et al, 2003; Luthi et a I, 2003; Zhang et a 1, 1994; Zha ng et a I, 1997} and Αβ oligomerization (Bagriantsev & Liebman, 2006}, but more recently models have been established to study Αβ₄2 toxicity ( atlack & Lindquist, 2011). These models have shown their va lidity as they demonstrated the importance of vesicular trafficking and endocytosis to maintain Α ₄2 cytotoxicity (D'Angelo et a I, 2013; Treusch et a I, 2011). One of the genes identified in genome-wide screenings using the Αβ₄2 yeast model is YAP1802, encoding for the yeast ortholog of human PICALM, a well-known risk factor for AD (Harold et al, 2009}. I n addition, the Tau protein has been intensively studied in yeast, and a model for tau-opathies has been developed in yeast (Van Leuven & Winderickx, 2002). When human three-repeat and four-repeat Tau is expressed in S. cerevisiae, key features like the hyperphosphorylation, conformational cha nge a nd self-assembly of Tau are faithfully recapitulated . Interestingly, these characteristics were found to be modu lated by yeast Tau kinases Mdsl and Pho85, respectively the orthologs of human kinases GSK-3 and cdkS (Vandebroek et al, 2006; Vandebroek et a I, 2005). Furthermore, the expression of clinical FTDP-17 (frontotemporal dementia and Parkinsonism linked to chromosome 17) Tau mutants in yeast led to the identification of specific phospho-epitopes that appear to be decisive for the loss of the physiological function of Tau as microtubule-binding protein and the induction of its self- assembly. The sa me study also reported that oxidative stress and mitochondrial dysfunction enhance Tau self-assembly independently of the Tau phosphorylation status (Vanhelmont et al, 2010). However, the sole expression of Tau in yeast fails to induce a significant growth defect.

There remains a need in the art for a reliable, accurate model for Αβ and tau toxicity. Disclosed herein is the surprising finding that Αβ a nd ta u show a synergistic toxicity in yeast. The toxic effects of Αβ and tau co-expression are greater than effects observed when Αβ is expressed alone in yeast, and are greater than the additive effect that Αβ and tau together might be expected to yield (based on the effect when either Αβ or tau is expressed alone). The yeast cells described herein model a synergistic effects of Αβ and tau, and provide a basis for elucidating the mechanisms of Αβ and tau toxicity, as well as for identifying compounds that reduce Αβ and tau toxicity. SUMMARY

One aspect of the present disclosure relates to a yeast cell com prising a first expression construct com prising a first promoter operably linked to a first n ucleic acid encoding a polypeptide comprising a secretion signal and an Αβ protein and a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding a polypeptide comprising a tau protein.

In some embodiments, the first promoter is a inducible promoter. The first promoter may be a GAL1 promoter, for exam ple, SEQ ID NO:52. in certain embodiments, the secretion signal is MFa prepro-leader sequence secretion signal (o* F). In some embodiments, the Αβ protein is a huma n Αβ peptide, for example, a human Αβ peptide selected from SEQ ID NO:l, SEQ 10 NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, a nd SEQ ID NO:6.

I n certa in embodiments, the human Αβ pe ptide is a mutant Αβ peptide, for example, Αβ 2(ΰ370) (SEQ ID NO:21).

In some embodiments, the human Αβ peptide is fused to gree fluoresce nt protein (G FP), for example, (SEQ ID IMO:35) to fo rm a human Αβ fusion protein. The human Αβ fusion protein may com prise a link between the human Αβ peptide and GFP. In some embodiments, the link is GAGAGA (SEQ ID NO:36), a nd the human Λβ fusion protein is ctMF-AR«.link-GFP (SEQ ID NO:2G) or a F^₂G37C-link-GFP (SEQ ID NO:28}.

In certain embodiments, the second promoter is a constitutive promoter. The constitutive promoter may be a GPD promoter, for example, (SEQ ID N0.53). tn some embodiments, the tau protein is a human tau protein. The human ta u protein may be isoform 2N/4R (SEQ I D NO:29). The human tau protein may be a mutant ta u protein, for example, a frontotemporal dementia linked to chromosome 17 (FTDP-17) mutant, in some embodiments, the ta u mutant is selected from SEQ ID NO: 30, SEQ ID NO:31, SEQ ID NO :32, SEQ ID NO:33, and SEQ ID NO:34. in certain embodiments, the yeast ceil is selected from Saccharomyces cerevisiae, Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomyces pombe, luyveromyces lactis, Hansenuia polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Y arrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., or Geotrichum fermentans. The yeast cell may be Saccharomyces cerevisiae. In some embodime nts, the yeast cell is a strain selected from BY4741 (Mat a his3Al leu2A0 metlSAO ura3A0) and the isogenic single or double deletion mutants mdslA, yapl801A, yapl802A, yapl801Ayapl802A, rvsl69A and sac6A.

In some embodiments, growth and/or viability of the yeast cell as described herein is reduced as compared to a control yeast cell. In some embodiments, the yeast ceil produces more reactive oxygen species than a control yeast cell. In some embodiments, cell necrosis is increased in the yeast cell as compared to a control yeast ceil. The control yeast cell may be a wild type yeast cell, a yeast cell expressing only Αβ, and a yeast cell expressing only tau. In some embodiments, tau phosphorylation in the yeast cell is increased as compared to a control yeast cell, wherein the control yeast cell is a yeast cell expressing tau but not Αβ. A further aspect of the present disclosure is the use of the yeast cell (any yeast cell described herein) as a model for cell toxicity mediated by activity of Αβ protein and tau. In certain embodiments, the use of the yeast cell is for screening com ounds for rescue of cell toxicity.

Yet another aspect of the present disclosure is a method for identifying a molecule which modifies at least one of Αβ toxicity, tau toxicity, or synergistic toxicity of Αβ and tau, said method comprising the steps of (a) providing a yeast cell as described herein; (b) contacting the yeast cell with the molecule; (c) determining effects of the molecule on the yeast cell; and (d) comparing the effects from step (c) with effects of the molecule on a control yeast cell, wherein a difference in the effects of step (c) and step (d) indicate that a molecule modifies at least one Αβ toxicity, tau toxicity, or the synergistic toxicity of human Αβ and human tau. In some embodiments, the control yeast cell is selected from a wild type yeast cell, a yeast ce!S expressing only Αβ, and a yeast cell expressing only tau. The control yeast cell may be grown in absence of the molecule.

In certain embodiments, the molecule is a small molecule compound or a biomolecule, for example, a protein, Another aspect of the present disclosure relates to the use of the yeast cell as described herein for obtaining hyperphosphorylated human tau. One aspect of the present disclosure relates to a method for obtaining purified hyperphosphorylated tau, comprising providing a yeast cell as disclosed herein, co-expressing both Αβ and tau in the yeast cell, and isolating purified hyperphosphorylated tau.

BRIEF DESCRIPTION OF DRAWINGS Fig. 1 shows growth profiles of BY4741 cells transformed with empty vector, a construct allowing for constitutive expression of protein Tau, constructs allowing for expression of A ₄₂Wt-GFP fusions with or without prepro a mating factor or combinations thereof. Growth was monitored in 96-multiwe!l plates using a Multiscan GO microplate spectrophotometer (Thermo Scientific).■ combined Tau and aA « t expression;♦ only αΑβ^ΐ expression;□ combined Tau and A ₄₂wt expression; O only A ₂Wt expression; · only Tau expression; O double empty vector control. Error bars represent the standard deviation of at least four independent transformants.

Fig. 2 shows growth profiles of BY4741 cells transformed with empty vectors, a construct allowing for expression of a wild type or mutant a F-Ap^-GFP fusion (αΑβ«νΛ, aA «G37C, αΑβ«ί34Τ), constructs allowing for constitutive expression of wild type Tau, or combinations thereof. Growth was monitored in 96-multiwell plates using a Multiscan GO microplate spectrophotometer (Thermo Scientific). ■ combined Αβ«νΛ and Tau expression; □ only aAp<2Wt expression; A combined αΑβ.·,2 ΐ.34Τ and Tau expression; Δ only αΑβ₄2ί34Τ expression; ♦ combined aA \cG37C and Tau expression; O only αΑβ.ΐ₂0370 expression; · only Tau expression; O double empty vector control. Error bars represent the SEM of at least four independent transformants.

Fig. 3 shows effects of MDSi-mediated hyperphosphorylation of Tau. Fig. 3A shows Western blot analysis of protein extracts from BY4741 and isogenic mdslA cells. Extracts of the left represent coexpression of aMF-Ap42Wt-GFP (aAP42Wt) and Tau. Extracts on the right represent expression of only Tau. Tau 5, Anti-GFP and Anti Adh2 were used as primary antibodies. Fig. 3B shows growth profiles of wild type BY4741 WT and isogenic mdslA cells transformed with empty vectors, a construct allowing for constitutive expression of protein Tau, a construct allowing for expression of a aMF-Ap₄₂Wt-GF fusion {aA 42Wt), or combinations thereof. Growth was monitored in 96-multiwell plate using a Multiscan GO microplate spectrophotometer (Thermo Scientific). Filled symbols represent 8Y4741 wild type cultures, open symbols represent mdslA cultures. ·, O control (twice empty vector); A, Δ cells with only Tau expression;♦, O cells with only o^₄?wt expression;■,□ cells with combined Tau and αΑβ₄2 expression. Error bars represent the SEM of at least four independent transformants. Fig. 4A shows Western blot analysis of protein extracts from BY4741 and isogenic mdslA celts. Three samples on the left represent coexpression of wild type aM F Ap₄2Wt~GFP (aA ₄2Wt) and Tau. Extracts on the right represent expression of only Tau. Primary antibodies used were Tau 5, AD2, ATS, AT180, AT270, 9G3 and ADH2. Fig. 4B shows relative immunoreactivity levels of Tau to different antibodies in protein extracts from BY4741 cells. All data are normalized for the total amount of Tau determined with the pan -Tau Mab auS. Filled bars represent relative immunoreactivity upon coexpression of Tau and aAP¾ t, open bars represent relative immunoreactivity upon expression of Tau alone. Error bars represent the standard deviation of at least three independent samples. Statistical analysis by t-test: n.s. P > 0,5; * P < 0,05; ** P < 0,01. Fig. 4C shows relative immunoreactivity levels of Tau to different antibodies in protein extracts from mdslA cells. All data are normalized for the total amount of Tau determined with the pan-Tau Mab Tau5. Filled bars represent relative immunoreactivity upon coexpression of Tau and aA ₄2Wt, open bars represent relative immunoreactivity upon expression of Tau alone. Error bars represent the standard deviation of at least three independent samples. Statistical analysis by t-test: n.s. P > 0,5; * P≤ 0,05; ** p < 0,01. No significant differences were observed.

Fig. 5 shows the effects of Tau mutants on cell growth. Fig. 5A shows growth profiles of BY4741 cells transformed with empty vectors, a construct allowing for expression of a ctMF-Ap^wt-GFP fusion (aAP«wt), constructs allowing for constitutive expression of wild type or mutant protein Tau (Tau, Tau-P301L, Tau-AK280), or combinations thereof. Growth was monitored in 96-multiwell plates as described above. ■ combined Tau wt and aAp ₂Wt expression; □ only Tau wt expression; A combined Tau P301L and aA3₄2Wt expression; Δ only Tau P301L expression;♦ combined Tau ΔΚ280 and ctA3«wt expression; O only Tau ΔΚ280 expression; · only aAfinwt expression; O double empty vector control. Error bars represent the SEM of at least 4 independent transformants. Fig. 5B shows a histogram representing the average V_max (AOD h) of the growth of BY4741 cells coexpressing ctAB₄2Wt and wild type or mutant Tau {Tau, Tau-P301L, Tau-AK280, Tau-G272V, Tau-V337M, Tau-R406W) as measured using a ultiscan GO microplate spectrophotometer (Thermo Scientific). Error bars represent the standard deviation of the V_ma)< of at least four independent transformants. Statistical analysis by t-test; * * P < 0,01. Coexpression of aAp^wt with Tau~P301L or Tau-AK280 showed a significantly lower Vmax when compared to coexpression of ctAp«wt and wild type Tau. All other combinations showed so significant difference. Fig. 5C shows a histogram representing the average Vmax of the growth of mdslA cells coexpressing ctAp₄2Wt and wild type or mutant Tau (Tau, Tau-P301L, Tau-AK280, Tau-G272V, Tau-V337M, Tau-R406W). No significant differences were observed. Error bars represent the standard deviation of the V_m3x of at least four independent transformants. Statistical analysis by t-test; coexpression of

and mutant Tau showed so significant (ns) differences in Vmax when compared to coexpression of <xAP_¾wt and wild type Tau.

Fig. 6 shows cells positive for fluorescence. Fig.6A shows a histogram representing the percentage of cells positive for DHE fluorescence. Results obtained after DHE staining of BY4741 cells expressing combinations of wild type or mutant MF-Ap42-GFP fusions ( AB₄2Wt, aAP4zG3 C, αΑβ42- 4Τ) and wild type Tau, or transformed with control plasmids after 24 hours of growth in medium containing 2% galactose. Filled bars represent coexpression αΑβ42 and Tau, open bars represent expression of αΑβ42 alone. Error bars represent the standard deviation of at least three independent transformants. Statistical analysis between strains with and without Tau by t-test: * P≤ 0,05; ** P < 0,01; *** P≤ 0,001. Fig. GB shows a fluorescent visualisation of DHE staining of BY4741 cells expressing combinations of wild type or mutant cxMF-Ap^-GFP fusions (αΑβ^νί, aAp_2G37C, aAp4jL34T) and wild type Tau, or transformed with control piasmids after 24 hours of growth in medium containing 2% galactose. Pictures on the left represent DHE fluorescence, pictures on the right represent a bright field image. Fig. 6C shows a histogram representing the percentage of cells positive for Pi fluorescence. Results obtained after PI staining of BY4741 cells expressing combinations of wild type or mutant ctMF- A 42-GF fusions (aAp«wt, aA ₄2G37C, αΑβ«-.34Τ) and wild type Tau, or transformed with control piasmids after 24 hours of growth in medium containing 2% galactose. Filled bars represent coexpression αΑβ and Tau, open bars represent expression of αΑβ alone. Error bars represent the standard deviation of at least three independent transformants. Statistical analysis between strains with and without Tau by t-test: * P≤ 0,05; ** P < 0,01; *** P < 0,001. Fig. 6D shows fluorescent visualisation of PI staining of BY4741 cells expressing combinations of wild type or mutant ojVlF-AB«- GFP fusions (uAp«wt, αΑβ«637(;, αΑβ«ί34Τ) and wild type Tau, or transformed with control piasmids after 24 hours of growth in medium containing 2% galactose. Pictures on the left represent Pi fluorescence, pictures on the right represent a bright field image.

Fig. 7 shows growth profiles of different yeast cells. Fig, 7A shows growth profiles of sac6A cells transformed with empty vectors, a construct allowing for expression of a wild type aMF-AP^wt-GFP fusion {aAp₄₂wt), a construct allowing for constitutive expression of wild type Tau, or combinations thereof. Growth was monitored in 96-multiwell plates using a Multiscan GO microplate spectrophotometer (Thermo Scientific). Filled symbols represent the BY4741 strains, open symbols represent the sac6A strains: ■, □ combined oAp₄2Wt a d Tau expression; ♦ , O only αΑ .^ Λ expression; A, Δ only Tau expression; ·, O double empty vector control. Frror bars represent the SEM of at least four independent transformants. Fig, 7B shows growth profiles of rvsl67A cells transformed with empty vectors, a construct allowing for expression of a wild type aM F^«wt-GFP fusion (aA ₄2Wt), a construct allowing for constitutive expression of wild type Tau, or combinations thereof. Growth was monitored in 96-multiwell plates as described above. Filled symbols represent the BY4741 strains, open symbols represent the rvsl67A strains:■,□ combined αΑβ«νΛ and Tau expression; O only otA$«wt expression; A, Δ only Tau expression; #, O double empty vector control. Error bars represent the SEM of at least four independent transformants. Fig. 7C shows growth profiles of BY4741 cells and the yaplSOlA yapl802A double deletion mutant transformed with empty vectors, a construct allowing for expression of a wild type

fusion (aA|3«wt), a construct allowing for constitutive expression of wild type protein Tau, or combinations thereof. Growth was monitored in 96-multiwell plates as described above. Filled symbols represent the BY4741 strains, open symbols represent the yaplSOlA γαρ1802Δ strains:■,□ combined Tau and cLA^wt expression;♦, O only αΑβ^νΛ expression; A, Δ only Tau expression; ·, O double empty vector control. Error bars represent the 5EM of at least four independent transformants.

DETAILED DESCRIPTION

This disclosure relates to yeast cells comprising transgenes encoding Αβ and tau, or mutants thereof. One aspect of the present disclosure relates to a yeast cell comprising (1) a first expression construct comprising a first promoter opera bly linked to a first nucleic acid encoding a polypeptide comprising a secretion signal and a human amyloid beta (Αβ) protein and (2) a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding a polypeptide comprising a human tau protein. In some embodiments, the nucleic acids encode polypeptides comprising Αβ or tau or mutants thereof. In certain embodiments, the yeast cells express Αβ and tau simultaneously. In some embodiments, the yeast cells express Αβ in a form that will aggregate in yeast. The yeast cells may express Αβ in a form that is or becomes cytotoxic to the cell, and causes a decrease in the growth and/or viability of the cell when expressed. In some embodiments, the co-expression of Αβ and tau leads to increased cytotoxicity and impairment of growth and/or viability of the yeast cells, as compared with expression of either Αβ or tau alone.The yeast cells may be used as models for cell toxicity, in particular, the synergistic toxicity conferred by the combination of Αβ and tau. The yeast cells may also be used to produce hyperphosphorylated tau.

Exemplary proteins and peptides of the present disclosure are described in SEQ ID NOS: 1-37, while exemplary nucleic acids which encode proteins and peptides or are promoter sequences of the present disclosure are described in SEQ ID NOS: 38-53.

Amyloid beta (Αβ) protein

Amlyoid-beta protein (also "Αβ ' "Abeta," or "beta amyloid") refers to peptides comprising 30-49 amino acids derived from the amyloid beta precursor protein (also "amyloid-β Precursor Protein ' "ΑβΡΡ," or "APP"). One exemplary form of APP is NP_000475 in the PubMed NCBI protein database (www.ncbi.nlm.nih.gov, version 19 Sept 2014). APP is cut by enzymes such as secretases, including alpha, beta, and gamma secretase, to produce Αβ oligomers. Soluble Αβ oligomers can exist in misfolded forms, some of which act as "seeds" to induce other Αβ molecules to misfold. Over time, misfolded Αβ oligomers can aggregate into insoluble fibrils, where they form the main component of protein aggregates known as amyloid plaques. Misfolded Αβ oligomers and the resulting amyloid plaques are highly toxic to cells, and are believed to play a role in amlyoidosis and neurodegenerative diseases such as Alzheimer's Disease. In humans, forms of Αβ protein (also referred to as Αβ peptides) include naturally occurring wiid type Αβ peptides as well as naturally occurring mutant Αβ peptides. Wild type Αβ peptides (also "Αβ wt") include forms such as Αβ residues 1-38 (Αβ₃₃) (SEQ ID NO:2), Αβ residues 1-39 (Αβ₃₉) (SEQ ID NO:3), Αβ residues 1-40 (Αβ₄ο) (SEQ ID NO:4), Αβ residues 1-41 (Λβ₄₁) (SEQ ID NO:5), Αβ residues 1-42 (Αβ,,₂) (SEQ ID NO:6), and Αβ residues 1-43 (Αβ«₃) (SEQ ID NO:l)of the sequence:

DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGWIAT (SEQ ID NO:l).

In some embodiments of the present disclosure, the Αβ protein is a human Αβ protein. Accordingly, where the yeast ceils described herein comprise a First expression construct comprising a first promoter operably linked to a first nucleic acid encoding a polypeptide comprising a secretion signal and an Λβ protein and a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding a polypeptide comprising a tau protein, the Αβ protein is a human Αβ protein. The protein may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to a human Αβ protein, in some embodiments, the Αβ protein is encoded by an ortholog of the human APP gene such as an APP gene from a non-human primate, rodent, canine, feline, or other animal.

The human Αβ may be wild-type Αβ. The yeast cell may comprise a nucleic acid encoding human Αβ in one or more different forms, for example the amyloid precursor protein (APP) or Λβ peptides such as Αβ₄₀ or Αβ₄₂. Αβ₄2 is more prone to aggregation and is more toxic. Studies suggest that the longer form Αβΐ2 plays a larger roie in the early stages of AD, and the balance between Αβ«ι and Αβ« tilts in favor of the longer form.

An Λβ protein may also be a mutant Αβ peptide, wherein the mutation occur in any of the forms of Αβ described in SEQ ID NOS: 1-6. For example, the Αβ peptide may abe the arctic mutant form (Αβ_Αρχ)· Alternatively, the Αβ may be a variant or mutant of a human Αβ peptide. A "variant human Αβ peptide" or a "mutant Αβ peptide" differs (via substitution, deletion, and/or insertion) from a naturally occurring Αβ peptide at up to 10 amino acids (e.g., differs at no more than 5 amino acids, differs at no more than 4 amino acids, differs at no more than 3 amino acids, differs at no more than 2 amino acids, or differs at 1 amino acid). A mutant Αβ peptide may be a clinical mutant, i.e., an Αβ peptide resulting from a copy of the APP protein that is encoded by a germ line mutation that underlies, or is associated with, Alzheimer's Disease. Exemplary Αβ mutations include but are not limited to A2T, HSR, D7N, A21 G, E22G (Arctic), E22Q (Dutch), E22K (Italian), D23N (Iowa), A42T, and A42V of the sequence:

DAEFRHDSGYEVHHQ LVFFAFDVGSNKGAIIGL VGGWIAT (SEQ ID NO: 1). The mutations may be in any form of Αβ peptide, as described above. Thus, the mutations above found in any one of SEQ ID NOS 1-6.

In some embodiments, the mutations in Αβ42 will have the sequences corresponding to SEQ ID NOS 7- 22. Additional exemplary Αβ mutants are E22K, A30V, !31T, G33V, L34T, G37C, and V40I. The mutants may vary in their toxic effects, for example, when the toxicity of Αβ 2 mutants are measured, G37C > A30V > G33V > E22K > V40I > E22G > wild type with respect to their ability to prevent growth and/or viability of cells (Vignaud et al., 2013). These mutations may be present Α 42 or in any of the Αβ peptides of SEQ ID NOS: 1-6. A polypeptide containing human Αβ protein may optionally be fused with a second domain. The second domain of the fusion protein can optionally be an immunoglobulin element, a dimerizing domain, a targeting domain, a stabilizing domain, or a purification domain. Alternatively, an Αβ protein can be fused with a heterologous molecule such as a detection protein. Exemplary detection proteins include: a fluorescent protein such as green fluorescent protein (GFP), cyan fluorescent protein (CFP) or yellow fluorescent protein (YFP); an enzyme such as β-galactosidase or alkaline phosphatase (AP); and an epitope such as glutathione-S-transferase (GST) or hemagglutinin (HA). To illustrate, an Αβ protein can be fused to GFP at the N- or C-terminus or other parts of the Αβ protein. These fusion proteins provide methods for rapid and easy detection and identification of the Αβ protein in the yeast cell. A Iinker sequence (also called "link") may be used to connect the Αβ peptide with a second domain such as GFP. The Iinker sequence may comprise small amino acids such as serine, glycine or alanine which separate Αβ from GFP. The iinker sequence prevents misfolding of GFP (and loss of fluorescence) upon Αβ aggregation. Such cis inactivation due to misfolding of the fusion partner is already described in Waldo et al. 1999). The length of the linker must provide enough flexibility and hydrophilicity to separate the two domains of the bi-functional protein. In some embodiments, the Iinker sequence is 6 amino acid residues, for example, GAGAGA (SEQ ID NO: 36). In certain embodiments, the Iinker is encoded by the nucleic acid GGTGCTGGCGCCGGTGCT (SEQ ID NO: 51).

In certain embodiments, the yeast cells described herein comprise a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding a polypeptide comprising a secretion signal and an Αβ protein, wherein the Αβ protein is an Αβ peptide (i.e., any of Α 3_, Α 39, Apw, Αβίΐ, Αβ₄₂, and Αβ₄₃, (SEQ ID NOS: 1-6) or any of the mutants described in SEQ ID NOS: 7-22) that has a Met residue at its N-terminus. Thus, the Αβ peptide is immediately preceded at its N-terminal end by a Met residue. Accord ingly, the Αβ peptide may be Met-Αβ, for example, Met-ApL^. An example of Αβ,₂ (SEQ !D NO: 6} in a construct which is preceded by Met is found in SEQ ID NO:23.

Exemplary embodiments of Αβ constructs, for example, comprising Αβ peptides that are Met-A _¾ peptides, are described in SEQ ID NOS: 23-28. (a refers to the mating factor a (MFa) prepro-leader sequence secretion signal).

Signal sequences and promoters

The Αβ protein enters the secretory pathway in order to exert its toxic effects. Thus, in some e mbodiments, the a signa l peptide such as a secretion signa l is expressed in frame with the Αβ protein. The expression construct of the yeast cells described herein may comprise a nucleic acid encoding a secretion signa l and a human a myloid beta protein. A "secretion signal" comprises a signal sequence pl us additional sequences required for efficient secretion of the polypeptide, for example, via processing in the Golgi apparatus. A "signal sequence" is a peptide sequence that is present within a polypeptide and causes the polypeptide to be targeted to the endoplasmic reticulum within a cell. A basic requirement for entry into the secretory pathway is an N-terminal sequence that d irects translocation into the endoplasmic reticulum ( ER), for example, a stretch of hydrophobic residues a nd a cleavage site recognized by signal peptidase in the ER lumen.

An exemplary secretion signal is S. cerevisiae pre-pro-a-factor. Pre-pro-a-factor (also called "mating factor ( F) a prepro-leader sequence secretion signal", " ", or "otM F") contains a signal seq uence of 19 amino acid residues terminating in a signal peptidase cleavage site, followed by a pro region of 66 residues, and a EAEA tetra peptide. In some embodiments, the yeast cells comprise an expression construct comprising a nucleic acid encoding a secretion signal that is the MFa prepro-leader sequence secretion signal. The nucleic acid may encode MFa prepro-leader sequence secretion signal and a huma n Αβ protein as described above.

In certain embodiments, the signal sequence is a Karp2 signal sequence or an Ostl signal sequence. To further promote processing and secretion of a non-yeast protein in a yeast cell, VpslO (a vacuolar sorting receptor) may be truncated, for example, by removal of its domain 1 (Fitzgera ld and Glick, 2014).

I n some embodiments, the signal sequence comprises KDEL (SEQ. ID NO :54) or HDEL (SEQ ID NO:55). This tetrapeptide motif directs proteins to the ER. The expression constructs disclosed herein may further comprise promoters, for example, a first promoter operably linked to a first nucleic acid encoding a polypeptide com prising a secretion signal and a human Αβ protein. A "promoter" is a control sequence that is a region of a nucleic acid sequence at which initiation and rate of transcription are controlled, it may contain genetic elements at which regulatory proteins and molecules may bind, such as RNA polymerase and other transcription factors, to initiate the specific transcription a nucleic acid sequence. The phrases "operatively linked" and "operatively positioned" mean that a promoter is in a correct functional location and/or orientation in relation to a nucleic acid sequence to control transcriptional initiation and/or expression of that sequence.

A promoter may be one naturally associated with a nucleic acid sequence, as may be obtained by isolating the 5' non-coding sequences located upstream of the coding segment and/or exon. Such a promoter can be referred to as "endogenous." Alternatively, a promoter may be a recombinant or heterologous promoter, which refers to a promoter that is not normally associated with a nucleic acid sequence in its natural environment. Such promoters may include promoters of other genes and promoters not "naturally occurring." The promoters employed may be either constitutive or inducible.

For example, various yeast-specific promoters (elements) may be employed to regulate the expression of a nucleic acid in yeast cells. Examples of inducible yeast promoters include but are not limited to CUP1, GALl-10, GAL1, GALL, GALS, TET, VP16 and VP16-ER. Examples of repressive yeast promoters include Met25. Examples of constitutive yeast promoters include g!yceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor- 1 -alpha promoter (TEF), cytochrome c-oxidase promoter (CYC1), and M P7. Autonomously replicating expression vectors of yeast containing promoters inducible by glucocorticoid hormones have also been described (Picard et a I, 1990), including the glucocorticoid responsive element (GRE). Still other yeast vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.

In some embodiments, a yeast strain is used that allows for expression, e.g., inducible expression, from GAL promoters on carbon sources otherthan galactose. In some embodiments, the strain carries an integrated or episomal (e.g., plasmid-borne) gene encoding a fusion protein, wherein the Gal4 DNA binding domain is fused to a transcriptional activation domain and a regulatory domain. The fusion protein is characterized in that its ability to activate transcription is regulated by binding of a small molecule to the regulatory domain. For example, in some embodiments, the fusion protein does not activate transcription in the absence of the small molecule, whereas in the presence of the small molecule, the fusion protein activates transcription. Exemplary small molecules include, e.g., steroid hormones, wherein the corresponding regulatory domain comprises at least a portion of a receptor for the small molecule. For example, the small molecule may be an estrogen (e.g., estradiol), or analog thereof (e.g., tamoxifen), and the corresponding regulatory domain comprises at least a portion of the estrogen receptor (ER). Exemplary activation domains inciude, e.g., viral protein activation domains such as the herpes simplex virus protein VP 16 activation domain. In some embodiments, the strain carries an integrated or episomai (e.g., plasmid-borne) gene encoding a Gal4 ER VP16 fusion protein. Presence of an estrogen receptor ligand, e.g., estradiol, in the medium, allows for expression from GAL promoters on carbon sources other than galactose. One of skill in the art will appreciate that numerous ways exist to render expression of a molecule of interest, e.g., an Αβ peptide, conditional, e.g., on culture media containing galactose or other carbon sources.

Yeast promoter sequences as described herein, as well as other constitutive or inducible promoters may be found in the Saccharomyces Genome Database at www.yeastgenome.org. Additional information on yeast promoters may be found in Johnston & Davis, 1984 and Bitter & Egan, 1984.

Exemplary promoter sequences are described in SEQ. ID NO:52 and SEQ ID NO:53.

Tau

Tau proteins (also "tau," "Tau," "τ," or "MAPT-encoded protein") are produced by alternative splicing from the single gene MAPT (microtubule-associated protein tau). The human MAPT gene is described in the NCBI gene database, version updated 3 Nov 2014, at Gene ID 4137. The tau proteins are derived from alternative mRNA splice variants that originate from a single MAPT gene and result in mature proteins that vary in size from 352 to 441 amino acids (36.8 to 45.9 kDa). There are six Tau isoforms, that differ from one another in having three or four microtubule binding repeats (R) of 31-32 amino acids each, and two, one or none amino terminal inserts (IM) of 29 amino acids each. The fetal brain contains a single isoform of tau (Tau-352) the adult brain has several isoforms all derived from a single gene by alternative mRNA splicing5.

Primarily expressed in neurons, tau proteins stabilize axonal microtubules while still permitting flexibility. These functions are dependent on the isoform of tau and the phosphorylation state of the isoform. While regulated phosphorylation of tau is essential for its function, hyperphosphorylation of tau (also "tau inclusions," or "pTau") has been implicated in tauopathies, such as frontotemporal dementia and Alzheimer's Disease. Insoluble aggregates of tau, including tangles of paired helical filaments and straight filaments, and neurofibrillary tangles ( FTs) are observed in neurons from patients with Alzheimer's Disease. Neuronal death of such cells is evident from the presence of "ghost" tangles, i.e. residues of dead tangle-bearing neurons in which essentially only the insoluble neurofibrillary tangles remain. Mechanisms of tau function and dysfunction are studied in numerous models, including yeast. Yeast cells which express human tau have been used as models for aspects of Alzheimer's Disease and other tauopathies (Van Leuven & Wtnderickx, 2002). These yeast are used to study processes such as formation of mitotic bundles, of pseudo-hyphen, of scar-sites, of cell-size, of cell-growth in defined conditions, of response to external signals, agent or compound.

As disclosed herein, the combined effects of Αβ and tau may also be studied in yeast cells. A first expression construct comprising a first promoter operably linked to a first nucleic acid encoding a polypeptide encoding a secretion signal and an Αβ protein is described above. A yeast celt comprising the first expression construct may further comprise a second expression construct comprising a second promoter operably linked to a second nucleic acid encoding a polypeptide comprising a tau protein. In some embodiments, the tau protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to a human tau protein. In some embodiments, the tau protein is an encoded by an ortholog of the human MAPT gene, for example, a tau protein is from a primate, rodent, canine, feline, or other animal. In some embodiments, the tau protein is a human tau protein. The human tau protein may be a wild- type tau protein, and may be one of the 6 human isoforms of tau. In the human brain, tau is expressed as 6 isoforms containing either 0, 1 or 2 N-terminal inserts and 3 or 4 microtubule binding repeats. In some embodiments, the human tau is selected from any one of the 6 isoforms. In certain embodiments, the tau is a 2N/4R isoform of tau (SEQ ID NO:29). Notably, the 2N/4R isoform is the longest isoform and yields more sarkosy!-insoluble Tau when expressed as hyperphosphorylated in yeast as com ared to the 2N/3 isoform (Vandebroek T et al., 2005). In some embodiments, the tau is a clinical tau mutant, i.e., a mutant observed in patients with tauopathies caused by mutations in tau. Many different mutations in APT have been observed in patients with frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). The tau protein may also be a synthetic mutant which is rapidly hyperphosphorylated. Exemplary tau mutants include, but are not limited to Tau 2N/4R (P301L) (SEQ (D NO:30); Tau 2N/4R (ΔΚ280) (SEQ ID NO:31); Tau 2N/4R (G272V) (SEQ ID NO:32); Tau 2N/4R (V337M) (SEQ ID NO:33); and Tau 2N/4R (R406W) (SEQ ID NO:34).

Co-expression of Αβ protein and tau protein: synergistic toxcity

A further aspect of the present disclosure is the synergistic activity of Αβ protein and tau protein in the yeast ceil in which both proteins are expressed. In some embodiments, a yeast cells as described herein is a model for synergistic effects of Αβ protein and tau protein, for example, for the synergistic cell toxicity mediated by the combined actions of Αβ protein and tau protein. In some embodiments, expression of the tau protein alone in a yeast cell does not cause cytotoxicity or other impairments in cell growth and/or viability, but co-expression of Αβ protein and tau protein in the yeast cell causes cytotoxicity and /or impairments in cell growth. Similarly, in certain embodiments, expression of the Αβ protein alone in a yeast cell is toxic, but co-expression of Αβ protein and tau protein in a yeast cell exacerbates the effects of Αβ protein. The effects of Αβ protein and tau protein together may be greater than the additive effect that Αβ and tau together might be expected to yield, based on the effect when either Αβ or tau is expressed alone. Thus, Αβ protein and tau protein may synergistically reduce viability and/or extend the time required to obtain colonies, as compared to Αβ alone.

Cytoxicity may be measured using cellular growth assays, for example by assessing colony formation or measuring OD600 of cell culture media. Generation of reactive oxygen species (ROS) is a response to environmental stress and may result in cell damage and cytotoxicity. Accordingly, in certain conditions, increased production of ROS also indicates cytotoxicity. in certain embodiments, co-expression of Αβ protein and tau protein in a yeast cell leads to increased tau phosphorylation as compared to a yeast celt in which tau protein is expressed alone. Co-expression of Αβ protein and tau protein in a yeast cell may also cause an increase in formation of oxygen radicals. Expression of Αβ« peptide alone causes an increase in reactive oxygen species (ROS), as quantified, for example, by measurement of DHE staining. Similarly, expression of tau protein alone causes an increase in ROS, although to a lesser extent than Αβ« peptide expression alone. However, co-expression of both Αβ« peptide and tau protein leads to a signtificantly larger increase in DHE fluorescences, greater than the sum of both separately. In some embodiments, co-expression of Αβ protein and tau protein leads to an increase in cell necrosis. Cell necrosis can be measured by propidium iodide (PI) staining. Co-expression of both Λβ« peptide and tau proteinleads to a significantly larger increase in eel! necrosis, as compared with either Αβ« peptide or tau protein expressed singly. In some embodiments, the expression constructs in the yeast cell are together on one plasmid. In some embodiments, the expression constructs are present on separate constructs. Αβ protein and tau protein may be expressed serially, or simultaneously, and expression of Αβ protein may be induced while expression of tau is constitutive, or vice versa.

A further aspect of the present disclosure relates to a yeast cell comprising a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding a polypeptide comprising a secretion signal and an Αβ protein and a second expression construct comprising a second promoter opera bly finked to a second nuc!eic acid encoding a polypeptide comprising a tau protein, wherein the Αβ protein is selected from SEQ ID NOS: 1-23, and the tau protein is selected from SEQ ID NO: 29-34.

In some embodiments, yeast cell comprises a first expression construct comprising a first promoter operab!y linked to a first nucleic acid encoding a polypeptide comprising a secretion signal and an Ap protein that is at least 50% homologous to any one of SEQ ID NOS: 1-22 and a second expression construct comprising a second promoter operably linked to a second nuc!eic acid encoding a polypeptide comprising a tau protein that is at least 50% homologous to any one of SEQ ID NOS: 29- 34. In some embodiments, the Αβ protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to any one of SEQ ID NOS: 1-22. In some embodiments, the tau protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to any one of SEQ ID NOS: 29-34.

In some embodiments, an Αβ protein {or Αβ fusion protein) in the left column is co-expressed with one of the tau proteins from the right coiumn. aMF refers to the MFa prepro-!eader sequence secretion signal.

α Γ-ApVG FP (SEQ ID NO:25) Tau V337 (SEQ ID NO:33) α Ρ-Λβ«·6ΡΡ (SEQ ID NO:25) Tau R406W(SEQ ID NO:34)

aM F-Ap₄∑- link-6FP (SEQ ID NO 26} Tau, 2 /4R isoform (SEQ ID NO :29)

aM F-A ink-GFP (SEQ ID NO 26) Tau P301L (SEQ ID NO:30)

aM F-Ap4?-link-GFP (SEQ ID O 26) Tau ΔΚ280 (SEQ ID NO:31)

aMF^«-link-GFP (SEQ ID NO 26) Ta u G272V (SEQ ID NO;32)

aMF-Ap«-!ink-GFP (SEQ SD NO 26) Tau V337M (SEQ ID O:33)

aMF-AP«-tink-GFP (SEQ iD NO 26) Tau R406W(SCQ ID NO: 4)

aM F-AP«(G37C)-link-GFP (SEQ ID NO 28) Tau, 2N/4R isoform (5EQ ID NO:29)

a F-Ap«(G37C)-link-GFP (SEQ I D NO 28) Tau P301L (SEQ !D NO:30)

aM F-Ap₄₂(G37CHink GFP (SEQ I D NO 28) Tau ΔΚ280 (SFQ ID NQ:31)

aMF-Ap 2(G37C)-Hnk-GFP (SEQ ID NO 28) Tau G272V (SEQ ID NO:32)

aMF-AP«(G37C) link G FP (SEQ ID NO 28) Tau 337M (SEQ ID NO:33)

F-AP42(G37C)-link-GFP (SEQ ID NO 28) Tau R406W(SEQ ID NO:34)

Yeast Cells

Yeast strains that can be used in the compositions and methods described herein include, but are not limited to, Saccharomyces cerevisiae, Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha, Pichia pastoris, Pichia methanolica, Pichia kluyveri, Yarrowia lipolytica, Candida sp., Candida utilis, Candida cacaof, Geotrichum sp., and Geotrichum fermentans. Although much of the discussion he rein relates to Saccharomyces cerevisiae which ectopica lly co-expresses Αβ a nd tau, this is merely for illustrative purposes. Other yeast strains can be substituted for S. cerevisiae. In some embodiments, the yeast cell is a strain BY4741 (Mat a his3Al leu2A0 metlBAQ ura3A0). Yeast cells as disclosed herein may be deletion mutants for genes involved in Αβ signaling pathways, tau signaling pathways, secretion, organization and/or maintenance of the actin cytoskeleton. For example, Sac6 deletion mutants or Rvsl67 mutants may be used because these proteins play a role in regulation, organization, and maintenance of the actin cytoskeleton. Co-expressing Αβ and tau in a yeast cell with such mutations may intensify the cytotoxic effects further. In some embodiments, the yeast cell is a isogenic single or double deletion mutants mdslA, yaplSOlA, yapl802A, ya p180l Ayapl802A, rvsl69A and sacSA.

Use of model

A further aspect of the present disclosure relates to the use of the yeast cell described herein as a model for ceil toxicity resulting from synergisitic effects of Αβ and tau. In some embodiments, the model is used to identify mechanisms and molecules that modify Λβ/tau-mediated cytotoxicity. In certain embodiments, the model is used to screen molecules capable of mod ifying Αβ/tau-mediated cytotoxicity. Molecules may exacerbate the synergistic toxicity of Αβ and tau, so that the deficits in growth and/or viability of yeast cells co-expressed with human Αβ and human tau are exacerbated. Molecules may lessen the synergistic toxicity of Αβ and tau, so that the deficits in growth and/or viability of yeast cells co-expressed with human Αβ and human tau are mitigated or lessened.

The mode! may comprise yeast cells of certain deletion strains, so that the growth defects mediated by Αβ and tau are strongest. In these embodiments, molecules which rescue the dramatic growth defects can be identified easily. For example, two deletion strains, sac6A and rvsl67A, each strongly increase the cytotoxic effect of Α 42. Then, upon co-expression of Α « and Tau, the growth of either of these deletion strains falls to almost zero. This almost zero-growth phenotype upon co-expression of Αβ« and Tau renders either sac6A or rvsl67A deletion strains suitable for screening compounds which reverse the near zero-growth phenotype. Similarly, where synergistic toxicity of Αβ₄2 and Tau is observed in deletion strains such as the yapl801A x apl802A deletion strain, this deletion strain may also be used in a screen for compounds that are suitable for reversing the phenotype. in some embodiments, the growth phenotype resulting from co-expression of Αβ₄₂ and Tau is almost zero growth, like the growth phenotype observed in sacSA and rvsl67A deletion mutants. In other deletion strains, the cytotoxicity does not lead to a severe, near zero-growth phenotype. Accordingly, a deletion strain may be selected for its growth phenotype when Αβ« and Tau are co-expressed, as a near zero-growth phenotype may be suitable for some compound screens whereas a wild-type growth phenotype may be best suited for other purposes.

In certain embodiments, a method for identifying a molecule which modifies the synergistic toxicity of human Αβ and human tau (for example, Αβ/tau-mediated cytotoxicity) comprises the steps of: (a) providing a yeast cell as disclosed herein, (b) contacting the yeast cell with the molecule, (c) determining the effect of the molecule on the yeast cell and comparing the effects from step (c) with effects of the molecule on a control yeast cell, wherein a difference in the effects of step (c) and step (d) indicate that a molecule modifies the synergistic toxicity of human Αβ and human tau. In some embodiments, the molecule exacerbates the synergistic toxicity, for example, leading to an increase in toxic effects such as cytotoxicity, generation of reactive oxygen species, cell necrosis, !n certain embodiments, the molecule mitigates the synergisitic toxicity, leading to a decrease in toxic effects such as cytotoxicity, generation of reactive oxygen species, and cell necrosis. In some embodiments, the control yeast cell is a wild type yeast cell. The control yeast cell may also be a yeast ceil of the same strain as the yeast cell, but not co-expressing Αβ and tau. For example, the control yeast cell may be a yeast cell that expresses only Αβ or only tau. The Αβ or tau that is singly expressed in a control yeast cell may be the equivalent peptide/protein of the Αβ and tau that are co-expressed together in the yeast cell. The control yeast cell may a lso be the identical yeast cell, grown in the absence of the molecule. in some em bodiments, synergistic toxicity of Αβ and tau is measured as cytotoxicity and a molecule may increase or decrease the cell toxicity mediated by Αβ and tau. The molecules may be small molecule compounds, biomolecules such as proteins or nucleic acids, or a combination of small molecules and biomolecules. Proteins which may increase or decrease Αβ/tau- mediated cytoxicity include but are not limited to heat shock proteins, proteins involved in oxidative stress responses, proteins involved in vesicular trafficking, proteins involved in cytoskeleton formation, mitochondrial proteins, and more. Yet a nother aspect of the present disclosure relates to the use of the yeast cells for o btaining hyperpho5phorylated tau. In certain embodiments, a method for obtaining hyperphosphorylated tau comprises providing a yeast cell as described herein and co-expressing both Αβ and tau in the yeast cell, wherein co-expression of Αβ and tau produces hyperphosphorylated tau. For example, the method produces hyperphosphorylated human tau species of a high molecular weight, such as 60 kDa or higherl n some embodiments, the hyperphosphorylated human ta u reaches a phosphorylation status different from that obtained when protein Tau is expressed aione in either wild-type yeast cells or cells lacking the Pho85 protein kinase, the ortholog of human CDK5. n some embodiments, the hyperphosphorylated human tau isolated from the yeast cells can be used as antigen for immu nization studies and the production of novel phospho-specific Tau antibodies or antibodies recognizing a specific Tau conformation or specific Tau oligomers. A method for isolation of heterologously expressed human protein Tau from yeast has been described previously (Vandebroek T et al. (2005).

EXAMPLES

The following are examples of the practice of the invention. They are not to be construed as limiting the scope of the invention in any way. a and MFa refer to the M F prepro-leader sequence secretion signal.

In intial experiments, the cytotoxic effects of d ifferent Αβ constructs co-expressed with tau constructs was determined. A cellular growth assay was first performed in BY4741 wild-type (wt) cells transformed with different constructs αΑβ wt-link-GFP, αΑβ arc-link-GFP, αΑβ 637C-link-GFP, or an URA control vector. Cytotoxic effects were then measured in cells that co-express Αβ a nd tau wt constructs (the combinations were Αβ wt-link-GFP + tau wt, αΑβ G37C-link-GFP + tau wt, URA control + ta u wt, or the αΑβ constructs with a HIS control vector). αΑβ constructs without GFP tags could a lso mediate cytotoxic effects in growth assays of BY4741 wt cells transfo rmed with αΑβ wt, αΑβ G37C, or a URA control vector, and ceil growth was measured. Co-expression of tau with these αΑβ constructs exacerbated the cytotoxic effect. Notably, neither Αβ-GFP nor Αβ-GFP + tau wt caused cytotoxicity, in contrast to αΑβ-GFP a nd αΑβ-GFP + tau wt, indicating the alpha mating factor and secretion signal necessary for secretory pathway targeting is also necessary for cytotoxicity. Measurements of dihydroethidium (DHE), an oxidizable fluorescent dye, demonstrated an increase in reactive oxygen species (ROS), which correlated with increased cytotoxicity of cells transformed with Αβ and tau (cells were transformed with αΑβ wt-Iink-GFP + tau wt or αΑβ G37C-iink-GFP + tau wt and compared with DHE fluorescence from cells that were transformed with αΑβ wt-Iink-GFP + HIS control or αΑβ G37C- link-GFP + tau wt). Protein expression was a lso examined in cells expressing Αβ and tau. BY4741 wt cells transformed with αΑβ wt-Iink-GFP + tau wt were compared with the protein expression of cells transformed with αΑβ wt-Iink-GFP + HIS control, and a control URA control vector + ta u wt or a control U RA control vector + HIS control. An anti-GFP antibody was used. Three bands corresponding to Αβ protein can be observed: (1) a glycosylated immature αΑβ-link-GFP, (2) unglycosylated immature αΑβ-link-GFP, and (3) a mature Αβ-link-GFP in which the ot-mating factor has been cleaved off. Tau did not affect Αβ expression levels. Conversely, expression of Αβ affects tau expression in several ways. Expression of ta u was measured using an anti-tau antibody in cells expressing αΑβ wt-Iink-GFP + ta u wt, αΑβ G37C- link-GFP + tau wt, or a URA control + ta u wt. Αβ expression over 6 hours has led to increased tau breakdown, emergence of higher molecular weight bands, and increased tau expression. After 24 hours of galactose ind uction of αΑβ. Here, the majority of tau can be found in the least mobile, most phosphorylated isoform. Additionally, in long induction experiments, hyperphosphorylation of tau was detected after 24 hours of Αβ induction. Cells were transformed with αΑβ wt-Iink-GFP + tau wt, αΑβ G37C-link-GFP + tau wt, or U RA control + tau wt, following 24 hours of galactose induction of Αβ, measured with the phosphospecific anti-tau antibody AT270 (which detects phosphorylation of T181). Phosphorylation of T231, S235, and Y18 was detected with the phosphospecific antibody AT180.

Example 1. Αβ₄₂ directed through the secretory pathway causes a synergistic growth defect with Tau in yeast

The exact molecular basis of the link between Αβ₄2 and Tau in Alzheimer's disease is still elusive. In order to address this issue, we engineered a novel yeast model and cotransformed the BY4741 wild yeast strain with a Gateway construct expressing human Tau (longest four-repeat isoform) from a constitutive GPD promoter, and a plasmid expressing an otMF-Ap«-GFP (also referred to as αΑβ₄2 νΐ) fusion protein from the galactose-inducible GAL1 promoter. It was already reported that this Αβ- fusion, which is processed through the secretory pathway by means of the alpha mating factor prepro leader sequence, is capa le of causing cytotoxicity when expressed in Soccharomyces cerevisiae (D'Angelo et a!, 2013}. Upon coexpression of this construct with the Tau protein, an even la rger decrease in growth could be observed. As Tau by itself does not cause a growth defect in S. cerevisiae, this result indicates that aAfinVJt and Ta u exert a synergistic cytotoxic effect (Figs.l & 2, Table I). To exclude marker-specific effects, we tested Tau vectors with HIS3 and LEU2 as auxotrophic ma rkers. In both systems, coexpression of aA ^wt and Ta u caused a simila r synergistic growth defect. In the following experiments, we continued with the HI S3 system.

!n order to verify that cytotoxicity of Αβ« is required to ind uce Tau toxicity, we coexpressed Tau with a non-toxic A 4 GFP construct (also referred to as Ap₄2-GFP). This construct is not preceded by the alpha mating factor prepro sequence, does not pass through the secretory pathway and does not cause a growth defect {D'Angelo et a I, 2013). In this case, Ta u fails to exert a synergistic growth inhibitory effect, thereby confirming the necessity of a toxic form of Αβ¾ to convert Tau into a cytotoxic entity (Fig. 1). Consequently, all following experiments were performed using alvlF-A «-GFP constructs. Next, we coexpressed Tau with two different Αβ« mutants, previously generated by random mutagenesis and selected by a ma rked increase or decrease in toxicity (Vignaud et a I, 2013). The aAP«G37C mutant shows a strong increase in toxicity, while the αΑβ_¾!34Τ muta nt only causes a margina l growth defect when expressed in yeast (Vignaud et a I, 2013). When Tau was expressed with the aAp₄;»G37C mutant in the BY4741 cells, growth was severely impaired, falling to almost zero. To our surprise, even when Tau was expressed in combinatio n with the αΑβ«ί34Τ muta nt a significant decrease in growth could still be observed, indicative that even a limited Αβ42 toxicity is sufficient to trigger the Tau synergistic effect (Fig. 2).

Example 2. Α « triggers dsl-mediated hyperphosphoryiation of protein Tau

As Αβ accumulation is known to lead to Tau hyperphosphoryiation in neurons of diseased brain (Blurton-Jones & Laferla, 2006), we analyzed the phosphorylation status of Tau in our yeast model. Detection of Tau using the pan-antibody Tau5 in protein extracts of BY4741 cells expressing only Tau showed a pattern of different bands with an apparent molecular weight between 64 and 72 kDa that correspond to different Tau phospho-variants as previously reported (Vandebroek et al, 2005; Va nheimont et al, 2010). In protein extracts obtained from BY4741 cells expressing Tau in combination with oj a clear shift towards the higher molecular weight bands is observed, indicative that Αβ,ζ triggers Tau hyperphosphoryiation also in yeast (Fig. 3A, Fig. 4A). This shift was even more prono unced when using a ntibodies directed towa rds specific phospho-epitopes, including AD2, ATS, AT180, AT270 and 9G3 antibodies (Table II, Fig. 4A). Quantification of the total signal intensity and normalization to the signal intensity obtained with the pan-antibody Tau5 revealed a significant intensity increase for AD2, ATS and AT180 (Fig. 4B). The Iatter was not due to loading differences since the signal intensities obtained with anti-Ad h2, which served as control, was similar in the protein extracts obtained from cells expressing either protein Tau alone or in combination with αΑβ^ννί. Notably, Tau did not affect the expression level of aA wt or vice versa. aAS 2Wt was detected as three distinct isoforms, representing the prepro precursor (41 kDa), the glycosylated precursor (50 kDa), and the mature form (34 kDa) as was previously reported (D'Angelo et al, 2013; Vignaud et a I, 2013). Also the ratio between these Αβ₄2 isoforms remains the same whether Tau is coexpressed or not (Fig. 3AJ. GS -3p is one of the best established human Tau kinases that was suggested to link Αβ« and Tau in diseased brain (Hernandez et a I, 2010; Hernandez et al, 2013; Uorens-Martin et a I, 2014), and we previously demonstrated that dsl, the ortholog human ΰ5Κ-3β, sustains Tau hyperphosphorylation in yeast (Vandebroek et al, 2006; Vandebroek et a I, 2005; Vanhelmont et al, 2010). Therefore, we also analyzed the Ap ₂-induced hyperphosphorylation of Tau in the mdslA deletion strain. Western blot analysis of protein samples obtained from these mdslA cells and subsequent signal quantification showed that aAp₄₂wt was no longer able to induce Tau hyperphosphorylation, indicative that Mdsl is indeed mediating the process as could be expected (Fig. 3A, Fig. 4A-C). Furthermore, growth analysis demonstrated a significant reduced capacity of Tau to exert its synergistic toxic effect in mdslA cells coexpressing aA ^wt, thereby providing a direct link between Mdsl-dependent hyperphosphorylation and cytotoxicity of Tau. Note, however, that the synergistic toxic effect of Tau is not completely abrogated in the mdslA background, indicative that there is at least one additional factor at play by which

converts Tau into a cytotoxic entity.

Example 3. Aggregation-prone clinical Tau mutants increase the synergistic growth defect

The discovery of different mutations in protein Tau that are associated with FTDP-17 implicated the protein in neuronal loss and dementia (Rademakers et a I, 2004; Sergeant et al, 2005). The effect of these mutations ranges from altering the ratio between three-repeat and four-repeat Tau by influencing splicing efficiency, over affecting the microtubule binding capacities of Tau or accelerating Tau self-assembly (Sergeant et al, 2005). We expressed a number of clinical FTDP-17 Tau mutants along with aAp₄₂wt, i.e. Tau-AK280, Tau-P301L, Tau-G272V, Tau V337M and Tau-R406W, all in the four-repeat Tau isoform. While all these Tau mutants have been shown to form neuronal inclusions in vivo (Sergeant et al, 2005; van Swieten et a I, 2007), in vitro studies documented that especially Tau- ΔΚ280 and Tau-P301L have a much higher propensity to form paired helical filaments than the other mutants and wild type Tau (Barghorn et al, 2000; von Bergen et al, 2001). In wild type yeast ceils, a!i clinical Tau mutants were capable of causing synergistic toxicity when coexpressed with αΑ Α-wt (Fig. 5 A-B; Tab!e II) but, intriguing!¹/, Tau-AK28Q and Tau-P301L triggered a much larger growth impairment than the other Tau mutants, which displayed a similar synergistic cytotoxicity as that obtained with wild type Tau (Fig. 5 A-B). Hence, it appears that the ability of protein Tau to inhibit growth is closely correlated with its propensity to aggregate. Next, we coexpressed the FTDP-17 Tau mutants and aA «wt in the mdslA deletion strain to confirm the necessity of Tau hyperphosphorylation. As expected, Tau Δ 280 and Tau-P301L lost their capacity for enhanced growth impairment in the mdslA background and all clinical Tau mutants affected growth similar as wild type Tau (Fig. SC.).

Example 4. Αβ_4- and Tau expression enhances ROS formation and plasma membrane disruption. Similar as in metazoan ceils, cell death in yeast is associated with the accumulation of reactive oxygen species (ROS) and eventually the disruption of the plasma membrane (Carmona-Gutierrez et a I, 2010). To analyze whether there is a correlation between the synergistic cytotoxicity observed with wild type and mutant αΑβ« and Tau and the appearance of these cell death markers, we performed stainings with dihydroethidium (DHE) and propidium iodide (Pi). In cells grown for 24 hours on gaiactose- containing medium, expression of aAp₄₂wt triggered enhanced DHE staining and thus the accumulation of ROS as compared to the empty vector control. Aiso the PI signal was significantly higher with cells expressing aA 42 t. As expected, the highly toxic Ap«G37C mutant showed a more severe increase for both the DHE and PI fluorescence, while the less toxic αΑβκ L34T mutant displayed DHE and PI levels comparable to the empty vector control. When Tau was coexpressed, the percentage of positively DHE and PI stained cells further increased when compared to cells only expressing the αΑ ₄2 constructs. Notably, the expression of Tau alone already triggered a small but consistent increase in DHE and PI staining (Fig. 6A-D).

Thus, the observed growth defects and cytotoxicity caused by expression of the different αΑβ« variants, either alone or combined with Tau closely correlated with the extent of both ROS accumulation and plasma membrane disruption.

Example 5. Synergistic Αβ₄₂ and Tau cytotoxicity involves endocytosis and cytoskeleton organization

Previous reports found clear links between Αβ₄2- induced cytotoxicity and endocytosis (D'Angeio et a I, 2013; Treusch et a I, 2011). in order to further validate this connection, we expressed

and Tau in strains lacking specific functions involved in endocytosis.

cytotoxicity was severely increased in strains carrying a deletion of SAC6 (Fig. 7A) or RVS167 (Fig, 7B). Sac6 is an actin-bundltng protein and ortholog of the human fimbrin tso forms T- and L-piastin that were recently shown to interact with the activated GTPase Rab5 (Adams et a 1, 1995; Hagiwara et at, 2011). Rvsl67 is a homolog of mammalian amphiphysin that interacts with actin as well and that functions in the internalization step of endocytosis (Lombardi & Riezman, 2001). When Tau was expressed alongside aAp₄₂wt in the sac6A or rvsl67A deletion strains, growth fell to nearly zero, which was not unexpected because of the severe phenotype already associated to the expression of ahfaiwt alone. We also coexpressed Tau and otAp₄₂wt in the yaplSOlA yapl802A double deletion strain. Yapl801 and Yapl802 are involved in clathrin cage assembly and they represent the orthologs of the well-established AD risk factor PICALM. Previously, it had been reported that deletion of YAP1801 and YAP1802 influenced the cytotoxic effect of Αβ₄₂ in S. cerevisiae (D'Angelo et a I, 2013; Treusch et a I, 2011). We could confirm and extend this result since also the synergistic toxicity of c Ap^wt and Tau was markedly reduced in the in yaplSOlA yapl802A double deletion strain as compared to the wild type strain (Fig. 7C).

MATERIALS & METHODS

Yeast strains and media

The Saccharomyces cerevisiae strains used in this study were BY4741 (Mat a his3M leu2AQ metlSAO ura3A0) and the isogenic single or double deletion mutants mdslA, yaplSOlA, yapl802A, yapl801Ayapl802A, rvsl69A and sacSA (see Tabe! III). Deletion of MDS1 was obtained by replacement of corresponding coding regions with PCR-derived Kan X cassettes as reported previously (Vanhelmont et al, 2010; Wach et a 1, 1994). The yaplSOlA, yapl802A, rvsl69A and sac6A single deletion strains were obtained from the genome-wide yeast deletion collection, and the yap!801Ayapl802A double deletion was created as reported previously by mating the single yaplSOlA and yap!802A strains and subsequent sporulation (D'Angeio et al, 2013).

The plasmid expressing aMF-Ap ₂-link-GFP from the GAL1 promoter was described previously (D'Angelo et al, 2013). The G37C and L34T mutants were created using random mutagenesis as reported in (Vignaud et al, 2013). Wild type and FTDP-17 Tau mutants, all in the 2N/4R isoform, were cloned into Gateway expression vectors using previously reported Tau plasmids as template (Vandebroek et af, 2006; Vandebroek et al, 2005; Vanhelmont et al, 2010). AttB regions were added to the Tau sequence by PCR and the subsequent sequence was cloned into a Gateway entry vector using BP recombination. Subsequent cloning into destination vectors was performed using LR recombination (Albert) et al, 2007).

Standard yeast transformation techniques were applied (Gietz et a I, 1992). Cells were grown at 30°C on synthetic media supplemented with 2% glucose, lacking the appropriate supplements for selection. For induction of Αβ₄₂ expression, cells were resuspended in synthetic medium with 2% galactose. Plasmids

The different Αβ« constructs were created in the lab of Prof. Dr. C. Culli n (University of Bordeaux). The constructs were inserted in a pYe vector, under control of a GAL promotor. The tau construct used was created in the host laboratory (Prof. Dr. J. inderickx, Lab of Functional Biology, KU Leuven). The tau gene is under control of a constitutive GPD promotor.

Growth curves and spot assays

G rowth in liquid medium was monitored by making growth curves. Stationary phase cells, grown on synthetic medium containing 2% glucose, were diluted to an GDsoo of 0,1 in synthetic med ium containing a 2% galactose concentration. OD_Soo was measured at regular time points for 2-4 days, until the cells reached stationary phase. Cells grown overnight on glucose were diluted to an OD600 of 0,1 in gaiactose-containing synthetic medium. G rowth of the cells was measured at 600 nm in 96-well plates every two hours for 72 to 140 hours using a Multisca n™ GO icroplate Spectrophotometer (Thermo Scientific™).

On solid medium, growth was monitored by spot assay, by making 10-fold serial dilutions of exponentia l cultures growing on synthetic medium conta ining 2% glucose, starting from a n O Deoo of 1. Samples of each dilution were spotted onto synthetic medium containing 2% galactose. Plates were incubated at 30°C for 2-3 days.

Immunoblot analysis

Cultures grown overnight on glucose were diluted to an OD600 of 0,5 in synthetic medium containing ga lactose and grown for 24 hours. Three OD units were harvested by centrifugation and protein extracts were prepared using the alkaline lysis method. Yeast cells were permeabilized with 500 μί of 0,185M NaOH a nd 0,2% of β-mercaptoethanol. After 10 minutes incubation on ice, TCA was added to a final concentration of 5%, followed by an additiona l 10 minute incubation on ice. Precipitates were collected by centrifugation for 5 minutes at 13000 g. Pellets were resuspended in 50 μΐ of sam ple buffer (4% sodium dodecyl sulfate, 0.1M Tris-HCI pH 6.8, 4 niM EDTA, 20% glycerol, 2% 2- mercaptoethanol, and 0.02% bromophenoi blue) and 25 μί of 1 Tris-Base. Samples were separated by standard SDS PAGE on 10% polyacrylamide gels and further analyzed using standard Western blotting techniques (Towbin et al, 1979). Antibodies used are listed in Table II. For αΑβ/ Wt-GFP detection we used both an anti-GFP and artti-Αβ» antibodies, for protein Tau a pan-antibody (Tau5) and several antibodies directed against Tau phospho-epitopes. We used HRP-conjugated seconda ry antibodies and the ECL method for detection and visualized the blots using a UVP Biospectrum^® Multispectral Imaging System. Relative immunoreactivity was determined by densitometry comparison (UVP VisionWorks^® LS analysis softwa re) and normalized for tota l Tau amounts as measured by pan-Tau a ntibody Tau-5.

DHE staining Cells were grown overnight on glucose-containing synthetic medium. The cultures were then diluted to an ODBOQ of 0,5 and grown on galactose-containing synthetic medium for 24 hrs. An ODeoo unit of 0,5 was pelleted down and resuspended in 250 pL of a 1/1000 dilution of DHE (2,5 mg/mL stock) in PBS (pH 0,7). After 10 minutes of incubation in the dark, cells were washed in PBS and the DHE signal was measured using a platereader ( Beckman Coulter^® DTX880 Multimode Detector).

Fluorescence microscopy

Tests for plasma membrane disruption (PI staining) as well as tests for the accumulation of ROS (DHE staining) have been described previously (Carmona-Gutierrez et a I, 2010) and were performed using flow cytometric and/or microscopic analysis. Cultures grown overnight on glucose-containing synthetic medium were diluted to an OD₆oo of 0,5 into ga lactose-containing synthetic medium and grown for 24 hours before sta ining and analysis. Flow cytometric analysis was performed after staining with PI or DH E. Staining solutions were added to a final concentration of 5 μΜ for PI and 5 pg/m! for DHE. The cells were then incubated for 30 minutes at 30°C and analyzed with a G uava easyCyte 8HT benchtop flow cytometer (Ivlillipore). Data were ana lyzed using Flowjo software. For all stainings, we also performed a fluorescent microscopical analysis of the cells using a Leica DM4000B microscope. At least 500 cells of each condition were inspected, both for protein localization and staining experiments.

TABLES

Table I: Relative OD_max, Ty₂, V_max (ΔΟΟ h) and g values for growth of BY4741 cells expressing empty vectors, ctAP zwt, wild type or mutant protein Ta u, and com binations thereof. Values are expressed as a pe rcentual value + standard deviation, relative to the respective value of the strain transformed with the URA and HIS empty vectors.

Expression of OD^ j_m (h) (AOD/h) g (h) oAp« + Tau 61 ± 10,8 226,5 ± 31,3 21,2 ± 5,1 379 ± 84,9 aAp42wt + Tau-P301L 34,9 ± 13,2 228,9 ± 43 9₃5 ± 4,8 672,9 ± 216,9 αΑβ₄₂τνί + Tau-AK280 27,1 ± 10,9 260,3 ± 31 ,4 6,1 ± 3,5 1153,7 ± 345,2 aAp«wt + Tau-G272V 63,9 ± 5,8 243,2 ±22,4 21,5 ±3,8 359,8 ±54,1 oAp + Tau-V337M 63,5 ±7,2 256,9 ±40,6 19,4 ±4,4 403,6 ± 109,9 aA ₄₂wt + Tau-R406W 57,3 ± 7,2 264 ±40 16,3 ±2,4 500,1 ± 102,4

URA + Tau 96,8 ±4,7 101,8 ±9,7 97,8 ±7,1 102,4 ±5,4

URA + Tau-P301L 96,2 ± 2,6 108,6 ±6,5 90,4 ±5,3 106,7 ±4,8

URA+Tau-AK280 94,4 ± 6 112,5 ±7,8 86,1 ±7,8 125,9 ± 12,3

URA + Tau-G272V 100,9 ±4,9 102,3 ±2,8 105,6 ±6,8 95,4 ±2,7

URA + Ta¾-V337M 99,4 ± 4,4 97,5 ±14 102,8 ±8,3 96,5 ±4,3

URA + Tau-R406W 98,4 ±3,7 95,4 ±16,3 103,7 ±6,3 97,9 ±4,6 aAp₂wt + HIS 86,5 ±4 171,6 ±14,3 41,9 ±3,9 232,7 ± 12,6

URA + HIS 100 ± 3,6 100 ±9,7 100 ±4,5 100 ±3,1

Table l!: List of antibodies used

Antibody Specificity Source

Tau5 Tau; aa218-225 BD Pharmingen (San Dief ;o, CA)(Porzig et al, 2007)

AD2 Tan; pSer396/pSer404 Biorad (Buee-Scherrer et al, 1996)

AT8 Tau; pSer202/pThr205 Innogenetics (Porzig et al, 2007)

AT 180 Tau; pThr231/pSer235 Imiogenetics (Whiteman et al, 2011)

AT270 Tau; pThr!81 Imiogenetics (Whiteman et al, 2011)

9G3 Tau; pTyrl8 Medimabs

-ADH2 ADH2 specific Millipore

Anti-GFP GFP specific Roche

Anti-Abeta Abeta specific Innogenetics

Goat-Anti-Mouse-HRP Anti-mouse mAb Biorad

Goai-Anti-Rabbit-HRP Anti-rabbit mAb Santa Cruz

Table Ml: List of strains used

Strain Genotype Reference

BY4741 MATa his3A0 leu2A0 metl5A0 ura3A0 Winzeler et al, 1999

MATa his3A0 leu2A0 metl5A0 ura3A0

mdslA Vandebroek et al.2005

YMR139W::KanMX4

MATa his3A0 Ieu2A0 metl5A0 ura3A0

sac6A Winzeler etal., 1 99

YDR129C::KanMX4 MATa his3A0 leu2A0 metl SAO ura3A0 rvsl67& Winzeler et al., 1999

YDR388W::KanMX4

MATa his3A0 leu2A0 ura3A0

ναρ1801Δγαρ1802Δ D'Angelo et al. 2013

YHR161C:: anMX4, YGR241C::KanMX4

T le IV: Summary of sequences

References

Adams AE, Shen W, Lin CS, Leavitt J, Matsudaira P (1995) Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin. Mot Cell Biol 15: 69-75

Alberti S, Gitler AD, Lindquist S (2007) A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae. Yeast 24: 913-919

Almeida CG, Takahashi RH, Gouras GK (2006) Beta-amyloid accumulation impairs multivesicular body sorting by inhibiting the ubiquitin-proteasome system../ Neurosci 26: 4277-4288

Bagriantsev S, Liebman S (2006) Modulation of Abeta42 low-n oligomerization using a novel yeast reporter system. BMC Biol 4: 32 Barghorn S, Zheng-Fischhofer Q, Ackmann M, Biernat J, von Bergen M, Mandeikow EM, Mandeikow E (2000) Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias. Biochemistry 39: 11714-11721

Bitter GA and Egan KM (19S4) Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate-dehydrogenase gene promoter. Gene 32(3):263-74. Blurton-Jones M, Laferia FM (2006) Pathways by which Abeta facilitates tau pathoiogy. Curr Alzheimer Res 3: 437-448

Boland B, Kumar A, Lee S, Piatt FM, Wegiel J, Yu WH, Nixon A (2008) Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease. J Neurosci 28: 6926-6937 Buee-Scherrer V, Condamines O, Mourton Gilles C, Jakes R, Goedert M, Pau B, Delacourte A (1996) AD2, a phosphorylation-dependent monoclonal antibody directed against tau proteins found in Alzheimer's disease. Brain Res Mot Brain Res 39: 79-88

Carmona-Gutierrez D, Eisenberg T, Buttner S, Meisinger C, Kroemer G, Madeo F (2010) Apoptosis in yeast: triggers, pathways, subroutines. Cell Death Differ 17: 763-773 Caspersen C, Wang N, Yao J, Sosunov A, Chen X, Lustbader JW, Xu HW, Stern D, McKhann G, Van SD (2005) Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer's disease. Faseb J 19: 2040-2041 D'Angelo F, Vignaud H, Di Martino J, Salin B, Devin A, Cullin C, Marchal C (2013) A yeast model for a myloid-beta aggregation exemplifies the role of membrane trafficking and PiCAL in cytotoxicity. Dis Model Mech 6: 206-216

Da Rocha Souto 3, Coma M, Perez-Nievas BG, Scotton TC, Siao M, Sa nchez-Ferrer P, Hashimoto T, Fan Z, Hudry E, Barroeta I, Sereno L, Rodriguez , Sanchez M B, Hyman BT, Gomez-lsla T (2012) Activation of glycogen synthase kinase-3 beta mediates beta-amyloid induced neuritic damage in Alzheimer's disease. Neurobiol Dis 45: 425-437

Edbauer D, Winkler E, Regula JT, Peso!d B, Steiner H, Haass C (2003} Reconstitution of gamma- secreiase activity. Nat Cell Biol 5: 486-488 Fitzgerald a nd Glick (2014) Secretion of a foreign protein from budding yeasts is e nhanced by cotranslationai translocation and by suppression of vacuolar targeting. Microb Cell Factories 2014, 13(1):125.

Fol e!l J, Cowan CM, Ubhi KK, Shiabh H, Newman TA, Shepherd D, Mudher A (2010) Abeta exacerbates the neuronal dysfunction caused by human ta u expression in a Drosophila model of Alzheimer's disease . Exp Neurol 223: 01-409

Fra nssens V, Bynens T, Van den Brande J, Vande rmeeren K, Verd uyckt M, Winderickx J (2013} The benefits of humanized yeast models to study Parkinson's disease. Oxid Med Ceil Longev 2013: 760629

Gietz D, St Jean A, Woods RA, Schiestl RH ( 1992) Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res 20: 1425 Ginsberg SD, Alldred MJ, Counts SE, Cataldo AM, Neve RL, Jiang Y, Wuu J, Chao MV, M ufson EJ, Nixon RA, Che S (2010) Microarray analysis of hippocampal CA1 neurons implicates early endosomal dysfunction during Alzheimer's disease progression. Biol Psychiatry 68: 885-893

Glenner GG, Wong CW (1984) Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascula r amyloid protein. Biochem Biophys Res Commun 120: 885- 890

Goedert M, Wischik CM, Crowther RA, Walker J E, lug A (1988) Cloning and seq uencing of the cDNA encoding a core protein of the paired helical filament of Alzheimer disease: identification as the microtubule-associated protein tau . Proc Natl Acad Sci U S A 85: 4051-4055

Gotz J, Deters N, Doldissen A, Bokha ri L, Ke Y, Wiesner A, Schonrock N, Ittner LM (2007) A decade of tau transgenic animal models and beyond. Brain Pathol 17: 91-103 Grundke-lqbal I, Iqba! K, Tung YC, Quinlan M, Wisniewski HM, Binder LI (1986) Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeietal pathology. Proc Natl Acad Sci U S A 83: 4913-4917

Hagiwara M, Shinomiya H, Kashihara M, Kobayashi K, Tadokoro T, Yamamoto Y (2011) Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian ceils. Biochem Biophys Res Commun 407: 615-619

Hardy J, Selkoe DJ (2002) The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297: 353-356

Hardy TA, Wu D, Roach PJ (1995) Novei Saccharomyces cerevisiae gene, MRK1, encoding a putative protein kinase with similarity to mammalian glycogen synthase kinase-3 and Drosophila Zeste- White3/Shaggy. Biochem Biophys Res Commun 208: 728-734

Harold D, Abraham R, Holiingworth P, Sims R, Gerrish A, Hamshere M L, Pahwa JS, Moskvina V, Dowzell K, Williams A, Jones N, Thomas C, Stretton A, Morgan AR, Lovestone S, Powell J, Proitsi P, Lupton MK, Brayne C, Rubinsztein DC, Gill M, Lawlor B, Lynch A, Morgan K, Brown KS, Passmore PA, Craig D, McGuinness B, Todd S, Holmes C, Mann D, Smith AD, Love S, Kehoe PG, Hardy J, Mead S, Fox N, Rossor M, Collinge J, Maier W, Jessen F, Schumann B, Heun R, van den Bussche H, Heuser I, Kornhuber J, Wiitfang J, Dichgans M, Frolich L, Hampel H, Hull M, Rujescu D, Goate AM, Kauwe JS, Cruchaga C, Nowotny P, Morris JC, Mayo K, Sleegers K, Bettens K, Engelborghs S, De Deyn PP, Van Broeckhoven C, Livingston G, Bass NJ, Gurling H, McQuillin A, Gwil!iam R, Deloukas P, Al-Chalabi A, Shaw CE, Tsoiaki M, Singleton AB, Guerreiro R, Muhleisen TW, Nothen MM, Moebus S, Jockel KH, Klopp N, Wichmann HE, Carrasquil!o MM, Pankratz VS, Younkin SG, Holmans PA, O'Donovan M, Owen MJ, Williams J (2009) Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease. Nat Genet 41: 1088-1093

Hernandez F, Gomez de Barreda E, Fuster-Matanzo A, Lucas JJ, Avila J (2010) GSK3: a possible link between beta amyloid peptide and tau protein. Exp Neurol 223: 322-325

Hernandez F, Lucas JJ, Avila J (2013) GSK3 and tau: two convergence points in Alzheimer's disease. J Atzheimers Dis 33 Suppl 1: S141-144

Ittner LM, Gotz J (2011) Amyloid-beta and tau--a toxic pas de deux in Alzheimer's disease. Nat Rev Neurosci 12: 65-72 Ittner LM, Ke YD, De!erue F, Bi M, Gladbach A, van Eersel J, Wolfing H, Chieng BC, Christie Mi, Napier IA, Eckert A, Staufenbiel M, Hardeman E, Gotz J (2010) Dendritic function of tau mediates amyloid- beta toxicity in Alzheimer's disease mouse models. Cell 142: 387-397

Johnston M and Davis RW (1984) Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae . Mol. Cell. Biol. 4(8): 1440.

Keil U, Hauptmann S, Bonert A, Scherping I, Eckert A, M uller WE (2006) Mitochondrial dysfunction induced by disease relevant Abeta PP a nd tau protein mutations. J Alzheimers Dis 9: 139-146

Lewis J, Dickson DW, Lin WL, Chishoim L, Corral A, Jones 6, Yen SH, Sahara N, Skipper L, Yager D, Eckman C, Hardy J, Hutton M, McGowan E (2001) Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science 293: 1487-1491

Llorens-Martin M, Jurado J, Hernandez F, Avila J (2014) GSK 3beta, a pivotal kinase in Alzheimer disease. Front Mol Neurosci 7: 46

Lombardi R, Riezman H (2001) Rvsl61p a nd Rvsl67p, the two yeast amphiphysin homoiogs, function together in vivo. J Biol Chem 276: 6016-6022 Luthi U, Schaerer-Brodbeck C, Tanner S, iddendorp O, Edler K, Ba rberis A (2003) Human beta- secretase activity in yeast detected by a novel celiular growth selection system. Biochim Biophys Acta 1620: 167-178

Mager WH, Winderickx J (2005) Yeast as a model for medical and medicinal research. Trends Pharmacol Sci 26: 265-273 Ma nczak M, Anekonda TS, Henson E, Park BS, Quirw J, Reddy PH (2006) M itochondria are a direct site of A beta accumulation in Alzheimer's disease neurons: implications for free radical generation and oxidative damage in disease progression. Hum Mol Genet 15: 1437-1449

Masters CL, Simms G, Weinman NA, Multha up G, McDonald BL, Beyreuther K (1985) Amyloid p!aq ue core protein in Alzheimer disease and Down syndrome. Proc Nat! Acad Sci U S A 82: 4245-4249 Matlack K and Lindquist S (2011) Yeast Ceils Expressing Amyloid Beta and Uses Therefor. WO 2011/088059.

Moussa CE, Fu Q, Kumar P, Shtifman A, topez J R, Allen PD, LaFerla F, Weinberg D, Magrane J, Apra hamia n T, Walsh K, Rosen KM, Querfurth HW (2006) Transgenic expression of beta-APP in fast- twitch skeletal m uscle leads to caicium dyshomeostasis and IBM-like pathology. Faseb J 20; 2165-2167 Oddo S, Caccamo A, Shepherd JD, Murphy MP, Golde TE, Kayed R, Metherate R, Mattson MP, Akbari Y, LaFerla FM (2003) Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction. Neuron 39: 409-421

Park H, Karrs Tl, Kim Y, Choi H, Gwon Y, Kim C, Koh JY, Jung Y (2012) Neuropathogenic role of adenylate kinase-1 in Abeta-mediated tau phosphorylation via AMPK and GSK3beta. Hum Mol Genet 21: 2725- 2737

Picard D, Khursheed 8, Garabedian MJ, Fortin MG, Lindquist S, Yamamoto KR (1990) Reduced levels of ftsp90 compromise steroid receptor action in vivo. Nature. 8;348(6297):166-8.

Porzig R, Singer D, Hoffmann R (2007) Epitope mapping of mAbs ATS and Tau5 directed against hyperphosphory!ated regions of the human tau protein. Biochem Biophys Res Commun 358: 644-649

Pruping K, Voigt A, Schuiz JB (2013) Drosophila melanogaster as a model organism for Alzheimer's disease. Mol Neurodegener 8: 35

Puziss JW, Hardy TA, Johnson RB, Roach PJ, Hieter P (1994) MDS1, a dosage suppressor of an mckl mutant, encodes a putative yeast homolog of glycogen synthase kinase 3. Mol Ceil Biol 14: 831-839 Rademakers R, Cruts M, van Broeckhoven C {2004) The role of tau (MAPT) in frontotemporal dementia and related tauopathies. Hum Mutat 24: 277-295

Rajendran L, Annaert W (2012) Membrane trafficking pathways in Alzheimer's disease. Traffic 13: 759- 770

Rapoport , Dawson HN, Binder LI, Vitek MP, Ferreira A (2002) Tau is essential to beta -amyloid- induced neurotoxicity. Proc Natl Acad Sci US A 99: 6364-6369

Rhein V, Song X, Wiesner A, Ittner LM, Baysang G, Meier F, Ozmen L, B!uethmann H, Drose S, Brandt U, Savaskan E, Czech C, Gotz J, Eckert A (2009) Amyloid-beta and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice. Proc Natl Acad Sci U S A 106: 20057-20062 Sereno L, Coma M, Rodriguez M, Sanchez-Ferrer P, Sanchez MB, Gich 1, Agulio JM, Perez M, Avila J, Guardia-Laguarta C, Clarimon J, Lleo A, Gomez-isla T (2009) A novel GSK-3beta inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo. Neurobioi Dis 35: 359-367

Sergeant N, Delacourte A, Buee L (2005) Tau protein as a differentia! biomarker of tauopathies. Biochim Biophys Acta 1739: 179-197 Sofola O, Kerr F, Rogers I, Kiliick R, Augustin H, Gandy C, Allen J, Hardy J, Lovestone S, Partridge L (2010) Inhibition of GSK-3 ameliorates Abeta pathology in an adult-onset Drosophiia model of Alzheimer's disease. PLoS Genet 6: el 001087

Terwel D, M uyllaert D, Dewachter I, Borghgraef P, Croes S, Devijver H, Van Leuven F (2008) Amyloid activates GSK-3beta to aggravate neuronal tauopathy in bigenic mice. Am J Pathol 172: 786-798

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76: 4350-4354

Treusch S, Hamamichi S, Goodman JL, Mat!ack KE, Chung CY, Baru V, Shulman J , Parrado A, Bevis BJ, Valastyan JS, Han H, Lindhagen-Pe rsson M, Reiman EM, Evans DA, Bennett DA, Olofsson A, DeJager PL, Ta nzi RE, Caldwell KA, Caldwell GA, Lindquist S (2011) Functional links between Abeta toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast. Science 334: 1241-1245

Umeda T, aekawa 5, Kimura T, Takashima A, Tomiyama T, Mori H (2014) Neurofibrillary tangle formation by introducing wild-type human tau into APP transgenic mice. Acta Neuropathol 117: 685- 698 Van Leuven F and Winderickx J (2002) Ta u opathy Model, WO2002/068663. van Swieten JC, B ronner IF, Arma ni A, Severijnen LA, Kam phorst W, Ravid R, Rizzu P, Willemsen R, Heutink P (2007) The DeltaK280 mutation in MAP tau favors exon 10 skipping in vivo. J Neuropathol Exp Neurol 66: 17-25

Vandebroek T, Terwel D, Vanhelmont T, Gysemans M, Va n Haesendonck C, Engelborghs Y, Winderickx J, Van Leuven F (2006) Microtubule binding a nd clustering of human Ta u-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5. J Biol Chem 281: 25388-25397

Vandebroek T, Vanhelmont T, Terwel D, Borghgraef P, Lemaire K, Snauwaett J, Wera S, Van Leuven F, Winderickx J (2005) Identification and isolation of a hyperphosphorylated, conformationaliy changed intermediate of human protein tau expressed in yeast. Biochemistry 44: 11466-11475

Vanhelmont T, Vandebroek T, De Vos A, Terwel D, Lemaire K, Ana ndha kumar J, Franssens V, Swinnen E, Van Le uven F, Winderickx J (2010) Serine-409 phosphorylation and oxidative damage define aggregation of human protein tau in yeast. FEMS Yeast Res 10: 992-1005

Vtgnaud H, Bobo C, Lascu i, Sorgjerd KM, Zako T, Maeda M, Salin B, Lecomte S, Cullin C (2013) A structure-toxicity study of Ass42 reveals a new anti-parallel aggregation pathway. PLoS One 8: e80262 von Bergen M, Barghorn S, Li L, Marx A_; Biernat J, Mandelkow EM, Mande!kow E (2001) Mutations or tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta structure. J Biol Chem 276: 48165-48174

Wach A, Brachat A, Pohlmann R, Philippsen P (1994) New heterologous modules for classical or PCR- based gene disruptions in Saccharomyces cerevisiae. Yeast 10: 1793-1808

Waldo GS, Standish BM, Berendzen J, Terwilliger TC. (1990) Rapid protein-folding assay using green fluorescent protein. Nature Biotechnol., 17:691-695

Whiteman IT, Minamide LS, Goh de L, Bamburg JR, Goidsbury C (2011) Rapid changes in phospho- MAP/tau epitopes during neuronal stress: cofilin-actin rods primarily recruit microtubule binding domain epitopes. PLoS One 6: e2087S

Winderickx J, Delay C, De Vos A, Klinger H, Pellens K, Vanheimont T, Van Leuven F, Zabrocki P (2008) Protein folding diseases and neurodegeneration: lessons learned from yeast. Biochim Biophys Acta 17S3: 1381-1395

Wittmann CW, Wszolek MF, Shulman JM, Salvaterra PM, Lewis J, Hutton M, Feany MB (2001) Tauopath in Drosophila: neurodegeneration without neurofibrillary tangles. Science 293: 711-714

Zhang H, Komano H, Fuller RS, Gandy SE, Frail DE (1994) Proteolytic processing and secretion of human beta-amyloid precursor protein in yeast. Evidence for a yeast secretase activity. J Biol Chem 269: 27799-27802

Zhang W, Espinoza D, Hines V, Innis M, Mehta P, Miller DL (1997) Characterization of beta-amyloid peptide precursor processing by the yeast Yap3 and Mkc7 proteases. Biochim Biophys Acta 1359: 110- 122

EQUIVALENTS

While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

We claim:

1. A yeast cell comprising a first expression construct comprising a first promoter operab!y linked to a first nucleic acid encoding a polypeptide comprising a secretion signal and an Αβ protein and a second expression construct comprising a second promoter operabiy linked to a second nucleic acid encoding a polypeptide comprising a tau protein.

2. The yeast cell of claim 1, wherein the first promoter is a inducible promoter.

3. The yeast cell of claim 1 or claim 2, wherein the first promoter is a 6AL1 promoter, for example, SEQ ID NO:52.

4. The yeast cell of any one of claims 1-3, wherein the secretion signal is MFa prepro-ieader sequence secretion signal (c* F).

5. The yeast cell of any one of claims 1-4, wherein the Αβ protein is a human Αβ peptide.

6. The yeast ceil of claim 5, wherein the human Αβ peptide is selected from SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

7. The yeast celi of claim 5 or claim 6, wherein the human Αβ peptide is a mutant Αβ peptide.

8. The yeast cell of claim 7, wherein the mutant Λβ peptide is A ₄?(G37C) (SEQ ID NO:21}.

9. The yeast celi of any one of claims 5-8, wherein the human Αβ peptide is fused to GFP (SEQ ID NO:35) to form a human Αβ fusion protein.

10. The yeast cell of claim 9, wherein the human Αβ fusion protein comprises a link between the human Αβ peptide and green fluorescent protein (GFP).

11. The yeast cell of claim 10, wherein the Sink is GAGAGA (SEQ ID NO:36).

12. The yeast cell of claim 11 or 12, wherein the human Αβ fusion protein is aMF-Afv.₂.link GFP (SEQ ID NO:26) or aMF-Ap«G37C-link-GFP (SEQ ID NO:28).

13. The yeast celi of any one of claims 1-12, wherein the second promoter is a constitutive promoter.

14, The yeast cell of ciaim 13, wherein the constitutive promoter is a GPD promoter, for example, (SEQ iD NO:53).

15. The yeast cell of any one of claims 1-14, wherein the tau protein is a human tau protein.

16. The yeast cell of claim 15, wherein the human tau protein is isoform 2N/4R (SEQ ID O:29).

17. The yeast cell of claim 15 or claim 16, wherein the human tau protein is a mutant tau protein.

18. The yeast cell of claim 17, wherein the mutant tau protein is a frontotem oral dementia linked to chromosome 17 (FTDP-17) mutant,

19. The yeast cell of claim 17 or claim 18, wherein the tau mutant is selected from SEQ ID NO;

30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34.

20. The yeast cell of any one of claims 1-19, wherein the yeast cell is selected from Saccharomyces cerevisiae, Saccharomyces uvae, Saccharomyces kluyveri, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenufa polymorpha, Pichia pastoris, Pichia methano!ica, Pichia kluyveri, Y arrowia lipolytica, Candida sp., Candida utilis, Candida cacaoi, Geotrichum sp., or Geotrichum fermentans.

21. The yeast cell of claim 20, wherein the yeast celi is Saccharomyces cerevisiae.

22. The yeast cell of claim 21, wherein the yeast eel! is a strain selected from BY4741 (Mat a his3Al ieu2A0 metlSAO ura3A0) and the isogenic single or double deletion mutants mdslA, yaplSOlA, yapl802A, yapl801Ayapl802A, rvsl69A and sac6A.

23. The yeast cell of any one of claims 1-22, wherein growth and/or viability of the yeast cell Is reduced as compared to a control yeast cell.

24. The yeast cell of any one of claims 1-23, wherein more reactive oxygen species are produced in the yeast cell than in a control yeast cell.

25. The yeast cell of any one of claims 1-24, wherein celt necrosis is increased in the yeast cell as compared to a control yeast cell.

26. The yeast cell of any one of claims 23-25, wherein the control yeast cell is selected from a wild type yeast cell, a yeast cell expressing only Αβ, and a yeast cell expressing only tau.

27. The yeast ceil of any one of claims 1-23, wherein tau phosphorylation is increased in the yeast cell as compared to a control yeast cell expressing tau but not Αβ.

28. The use of the yeast cell of any one of claims 1-27 as a model for cell toxicity mediated by activity of Αβ protein and tau.

29. The use of claim 28 or 29 for screening compounds for rescue of cell toxicity.

30. A method for identifying a molecule which modifies at least one of human Αβ toxicity, tau toxicity, or synergistic toxicity of Αβ and tau, said method comprising the steps of:

a) providing a yeast cell of any one of claims 1-27,

b) contacting the yeast cell with the molecule,

c) determining effects of the molecule on the yeast cell, and

d) comparing the effects from step (c) with effects of the molecule on a control yeast cell, wherein a difference in the effects of step (c) and step (d) indicate that a molecule modifies at least one of human Αβ toxicity, tau toxicity, or the synergistic toxicity of human Αβ and human tau.

31. The method of claim 31, wherein the control yeast cell is selected from a wild type yeast cell, a yeast cell expressing only Αβ, and a yeast cell expressing only tau.

32. The method of cla im 30 or claim 31, wherein the control yeast celi is grown in absence of the molecule.

33. The method of claim 32, wherein the molecule is a small molecule compound or a biomolecule.

34. The method of claim 33, wherein the biomolecule is a protein.

35. The use of the yeast cell of any one of claims 1-27 for obtaining hyperphosphorylated human tau.

36. A method for obtaining purified hyperphosphorylated tau, comprising provid ing a yeast cell of any one of claims 1-27, co-expressing both Αβ and tau in the yeast celt, and isolating purified hyperphosphorylated tau .