WO2015066776A2 - Modèle de levure pour une toxicité synergique - Google Patents

Modèle de levure pour une toxicité synergique Download PDF

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WO2015066776A2
WO2015066776A2 PCT/BE2014/000062 BE2014000062W WO2015066776A2 WO 2015066776 A2 WO2015066776 A2 WO 2015066776A2 BE 2014000062 W BE2014000062 W BE 2014000062W WO 2015066776 A2 WO2015066776 A2 WO 2015066776A2
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tau
yeast cell
yeast
protein
seq
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PCT/BE2014/000062
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WO2015066776A3 (fr
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Christophe Cullin
Mathias VERDUYCKT
Hélène VIGNAUD
Joris Winderickx
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Katholieke Universiteit Leuven Ku Leuven Research & Development
Universite de Bordeaux
Centre National De La Recherche Scientifique
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/036—Fusion polypeptide containing a localisation/targetting motif targeting to the medium outside of the cell, e.g. type III secretion
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • This invention relates to yeast cells expressing ⁇ and tau.
  • the yeast DCis are suitable for analysis of various aspects of ⁇ and tau pathology, such as toxicity mechanisms underlying pathologies, identification of molecules which reduce toxic effects of ⁇ and tau, and formation of hyperphosphorylated tau species.
  • AD ⁇ Alzheimer's Disease
  • amyloid cascade theory where an increased generation of ⁇ 42 , 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 Ca 2 * homeostasis (Moussa et al, 2006).
  • 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).
  • AD amyloid precursor protein
  • the first promoter is a inducible promoter.
  • the first promoter may be a GAL1 promoter, for exam ple, SEQ ID NO:52.
  • the secretion signal is MFa prepro-leader sequence secretion signal (o* F).
  • the ⁇ protein is a huma n ⁇ peptide, for example, a human ⁇ 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.
  • the human ⁇ pe ptide is a mutant ⁇ peptide, for example, ⁇ 2( ⁇ 370) (SEQ ID NO:21).
  • the human ⁇ peptide is fused to gree fluoresce nt protein (G FP), for example, (SEQ ID IMO:35) to fo rm a human ⁇ fusion protein.
  • G FP gree fluoresce nt protein
  • the human ⁇ fusion protein may com prise a link between the human ⁇ peptide and GFP.
  • the link is GAGAGA (SEQ ID NO:36)
  • a nd the human ⁇ fusion protein is ctMF-AR «.link-GFP (SEQ ID NO:2G) or a F ⁇ 2 G37C-link-GFP (SEQ ID NO:28 ⁇ .
  • the second promoter is a constitutive promoter.
  • the constitutive promoter may be a GPD promoter, for example, (SEQ ID N0.53).
  • 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.
  • 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.
  • 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.
  • growth and/or viability of the yeast cell as described herein is reduced as compared to a control yeast cell.
  • the yeast ceil produces more reactive oxygen species than a control yeast cell.
  • 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 ⁇ , and a yeast cell expressing only tau.
  • 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 ⁇ .
  • Yet another aspect of the present disclosure is a method for identifying a molecule which modifies at least one of ⁇ toxicity, tau toxicity, or synergistic toxicity of ⁇ 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 ⁇ toxicity, tau toxicity, or the synergistic toxicity of human ⁇ and human tau.
  • the control yeast cell is selected from a wild type yeast cell, a yeast ce!S expressing only ⁇ , and a yeast cell expressing only tau. The control yeast cell may be grown in absence of the molecule.
  • 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 42 Wt-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 ⁇ expression; ⁇ combined Tau and A 42 wt expression; O only A 2 Wt expression; ⁇ only Tau expression; O double empty vector control. Error bars represent the standard deviation 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 Ap 4 2Wt ⁇ GFP (aA 4 2Wt) 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 aAP3 ⁇ 4 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 4 2Wt, 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.
  • 5B shows a histogram representing the average V max (AOD h) of the growth of BY4741 cells coexpressing ctAB 4 2Wt 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.
  • FIG. 5C shows a histogram representing the average Vmax of the growth of mdslA cells coexpressing ctAp 4 2Wt 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 m3 x 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 3 ⁇ 4 wt 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.
  • Filled bars represent coexpression ⁇ 42 and Tau, open bars represent expression of ⁇ 42 alone.
  • Error bars represent the standard deviation of at least three independent transformants.
  • Fig. GB shows a fluorescent visualisation of DHE staining of BY4741 cells expressing combinations of wild type or mutant cxMF-Ap ⁇ -GFP fusions ( ⁇ , 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.
  • 6D shows fluorescent visualisation of PI staining of BY4741 cells expressing combinations of wild type or mutant ojVlF-AB «- GFP fusions (uAp «wt, ⁇ «637(;, ⁇ « ⁇ 34 ⁇ ) 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 ⁇ aAp 42 wt), 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 oAp 4 2Wt a d Tau expression; ⁇ , O only ⁇ . ⁇ ⁇ expression; A, ⁇ only Tau expression; ⁇ , O double empty vector control.
  • 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
  • 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.
  • Amlyoid-beta protein refers to peptides comprising 30-49 amino acids derived from the amyloid beta precursor protein (also “amyloid- ⁇ Precursor Protein ' " ⁇ ,” or "APP").
  • amyloid beta precursor protein also “amyloid- ⁇ Precursor Protein ' " ⁇ ,” or "APP”
  • APP amyloid- ⁇ Precursor Protein '
  • 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 ⁇ oligomers.
  • Soluble ⁇ oligomers can exist in misfolded forms, some of which act as “seeds” to induce other ⁇ molecules to misfold. Over time, misfolded ⁇ oligomers can aggregate into insoluble fibrils, where they form the main component of protein aggregates known as amyloid plaques. Misfolded ⁇ 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 ⁇ protein (also referred to as ⁇ peptides) include naturally occurring wiid type ⁇ peptides as well as naturally occurring mutant ⁇ peptides.
  • Wild type ⁇ peptides include forms such as ⁇ residues 1-38 ( ⁇ 33 ) (SEQ ID NO:2), ⁇ residues 1-39 ( ⁇ 39 ) (SEQ ID NO:3), ⁇ residues 1-40 ( ⁇ 4 ⁇ ) (SEQ ID NO:4), ⁇ residues 1-41 ( ⁇ 41 ) (SEQ ID NO:5), ⁇ residues 1-42 ( ⁇ ,, 2 ) (SEQ ID NO:6), and ⁇ residues 1-43 ( ⁇ « 3 ) (SEQ ID NO:l)of the sequence:
  • the ⁇ protein is a human ⁇ 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 ⁇ 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 ⁇ protein is a human ⁇ protein.
  • the protein may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to a human ⁇ protein, in some embodiments, the ⁇ 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 ⁇ may be wild-type ⁇ .
  • the yeast cell may comprise a nucleic acid encoding human ⁇ in one or more different forms, for example the amyloid precursor protein (APP) or ⁇ peptides such as ⁇ 40 or ⁇ 42 .
  • APP amyloid precursor protein
  • ⁇ peptides such as ⁇ 40 or ⁇ 42 .
  • ⁇ 4 2 is more prone to aggregation and is more toxic.
  • An ⁇ protein may also be a mutant ⁇ peptide, wherein the mutation occur in any of the forms of ⁇ described in SEQ ID NOS: 1-6.
  • the ⁇ peptide may abe the arctic mutant form ( ⁇ ⁇ ⁇ ) ⁇
  • the ⁇ may be a variant or mutant of a human ⁇ peptide.
  • a "variant human ⁇ peptide” or a “mutant ⁇ peptide” differs (via substitution, deletion, and/or insertion) from a naturally occurring ⁇ 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 ⁇ peptide may be a clinical mutant, i.e., an ⁇ 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 ⁇ 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 ⁇ peptide, as described above. Thus, the mutations above found in any one of SEQ ID NOS 1-6.
  • the mutations in ⁇ 42 will have the sequences corresponding to SEQ ID NOS 7- 22.
  • Additional exemplary ⁇ mutants are E22K, A30V, !31T, G33V, L34T, G37C, and V40I.
  • the mutants may vary in their toxic effects, for example, when the toxicity of ⁇ 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 ⁇ 42 or in any of the ⁇ peptides of SEQ ID NOS: 1-6.
  • a polypeptide containing human ⁇ 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.
  • an ⁇ 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 ⁇ -galactosidase or alkaline phosphatase (AP); and an epitope such as glutathione-S-transferase (GST) or hemagglutinin (HA).
  • an ⁇ protein can be fused to GFP at the N- or C-terminus or other parts of the ⁇ protein.
  • These fusion proteins provide methods for rapid and easy detection and identification of the ⁇ protein in the yeast cell.
  • a Iinker sequence (also called "link") may be used to connect the ⁇ peptide with a second domain such as GFP.
  • the Iinker sequence may comprise small amino acids such as serine, glycine or alanine which separate ⁇ from GFP.
  • the iinker sequence prevents misfolding of GFP (and loss of fluorescence) upon ⁇ aggregation. Such cis inactivation due to misfolding of the fusion partner is already described in Waldo et al. 1999).
  • the Iinker sequence is 6 amino acid residues, for example, GAGAGA (SEQ ID NO: 36).
  • the Iinker is encoded by the nucleic acid GGTGCTGGCGCCGGTGCT (SEQ ID NO: 51).
  • 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 ⁇ protein, wherein the ⁇ protein is an ⁇ peptide (i.e., any of ⁇ 3_, ⁇ 39, Apw, ⁇ , ⁇ 42 , and ⁇ 43 , (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.
  • the ⁇ peptide is immediately preceded at its N-terminal end by a Met residue.
  • the ⁇ peptide may be Met- ⁇ , for example, Met-ApL ⁇ .
  • Met-ApL ⁇ Met-ApL ⁇ .
  • ⁇ constructs for example, comprising ⁇ peptides that are Met-A 3 ⁇ 4 peptides, are described in SEQ ID NOS: 23-28.
  • a refers to the mating factor a (MFa) prepro-leader sequence secretion signal.
  • Pre-pro-a-factor also called “mating factor ( F) a prepro-leader sequence secretion signal", " ", or "otM F"
  • F mating factor
  • 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 ⁇ protein as described above.
  • the signal sequence is a Karp2 signal sequence or an Ostl signal sequence.
  • VpslO a vacuolar sorting receptor
  • VpslO may be truncated, for example, by removal of its domain 1 (Fitzgera ld and Glick, 2014).
  • 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 ⁇ 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.”
  • 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.
  • yeast vectors 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.
  • 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.
  • 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.
  • 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.
  • an estrogen receptor ligand e.g., estradiol
  • Presence of an estrogen receptor ligand, e.g., estradiol, in the medium allows for expression from GAL promoters on carbon sources other than galactose.
  • an estrogen receptor ligand e.g., estradiol
  • 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.
  • 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.
  • 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.
  • a first expression construct comprising a first promoter operably linked to a first nucleic acid encoding a polypeptide encoding a secretion signal and an ⁇ 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.
  • the tau protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to a human tau protein.
  • 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.
  • 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.
  • the human tau is selected from any one of the 6 isoforms.
  • the tau is a 2N/4R isoform of tau (SEQ ID NO:29).
  • Exemplary tau mutants include, but are not limited to Tau 2N/4R (P301L) (SEQ (D NO:30); Tau 2N/4R ( ⁇ 280) (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).
  • a further aspect of the present disclosure is the synergistic activity of ⁇ protein and tau protein in the yeast ceil in which both proteins are expressed.
  • a yeast cells as described herein is a model for synergistic effects of ⁇ protein and tau protein, for example, for the synergistic cell toxicity mediated by the combined actions of ⁇ protein and tau protein.
  • 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 ⁇ protein and tau protein in the yeast cell causes cytotoxicity and /or impairments in cell growth.
  • ⁇ protein and tau protein may synergistically reduce viability and/or extend the time required to obtain colonies, as compared to ⁇ 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.
  • co-expression of ⁇ 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 ⁇ protein and tau protein in a yeast cell may also cause an increase in formation of oxygen radicals. Expression of ⁇ « peptide alone causes an increase in reactive oxygen species (ROS), as quantified, for example, by measurement of DHE staining.
  • ROS reactive oxygen species
  • the expression constructs in the yeast cell are together on one plasmid. In some embodiments, the expression constructs are present on separate constructs. ⁇ protein and tau protein may be expressed serially, or simultaneously, and expression of ⁇ 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 ⁇ 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 ⁇ protein is selected from SEQ ID NOS: 1-23, and the tau protein is selected from SEQ ID NO: 29-34.
  • 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.
  • the ⁇ protein is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% homologous to any one of SEQ ID NOS: 1-22.
  • 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.
  • an ⁇ protein ⁇ or ⁇ 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.
  • aM F-Ap 4 ⁇ - link-6FP (SEQ ID NO 26 ⁇ Tau, 2 /4R isoform (SEQ ID NO :29)
  • 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 ⁇ and tau in a yeast cell with such mutations may intensify the cytotoxic effects further.
  • the yeast cell is a isogenic single or double deletion mutants mdslA, yaplSOlA, yapl802A, ya p180l Ayapl802A, rvsl69A and sacSA.
  • a method for identifying a molecule which modifies the synergistic toxicity of human ⁇ and human tau 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 ⁇ and human tau.
  • the ⁇ or tau that is singly expressed in a control yeast cell may be the equivalent peptide/protein of the ⁇ 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 ⁇ and tau is measured as cytotoxicity and a molecule may increase or decrease the cell toxicity mediated by ⁇ 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 ⁇ /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.
  • a method for obtaining hyperphosphorylated tau comprises providing a yeast cell as described herein and co-expressing both ⁇ and tau in the yeast cell, wherein co-expression of ⁇ and tau produces hyperphosphorylated tau.
  • the method produces hyperphosphorylated human tau species of a high molecular weight, such as 60 kDa or higherl
  • 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.
  • 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).
  • a and MFa refer to the M F prepro-leader sequence secretion signal.
  • cytotoxic effects of d ifferent ⁇ 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 ⁇ wt-link-GFP, ⁇ arc-link-GFP, ⁇ 637C-link-GFP, or an URA control vector. Cytotoxic effects were then measured in cells that co-express ⁇ a nd tau wt constructs (the combinations were ⁇ wt-link-GFP + tau wt, ⁇ G37C-link-GFP + tau wt, URA control + ta u wt, or the ⁇ constructs with a HIS control vector).
  • ⁇ constructs without GFP tags could a lso mediate cytotoxic effects in growth assays of BY4741 wt cells transfo rmed with ⁇ wt, ⁇ G37C, or a URA control vector, and ceil growth was measured. Co-expression of tau with these ⁇ constructs exacerbated the cytotoxic effect. Notably, neither ⁇ -GFP nor ⁇ -GFP + tau wt caused cytotoxicity, in contrast to ⁇ -GFP a nd ⁇ -GFP + tau wt, indicating the alpha mating factor and secretion signal necessary for secretory pathway targeting is also necessary for cytotoxicity.
  • Cells were transformed with ⁇ wt-Iink-GFP + tau wt, ⁇ G37C-link-GFP + tau wt, or U RA control + tau wt, following 24 hours of galactose induction of ⁇ , 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 4 ⁇ 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 ⁇ « 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 aAp 42 wt triggered enhanced DHE staining and thus the accumulation of ROS as compared to the empty vector control.
  • DHE dihydroethidium
  • Pi propidium iodide
  • 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).
  • Tau was expressed alongside aAp 42 wt 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.
  • Yapl801 and Yapl802 are involved in clathrin cage assembly and they represent the orthologs of the well-established AD risk factor PICALM.
  • PICALM AD risk factor
  • 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).
  • the different ⁇ « 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.
  • 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 S oo 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 nTM GO icroplate Spectrophotometer (Thermo ScientificTM).
  • Yeast cells were permeabilized with 500 ⁇ of 0,185M NaOH a nd 0,2% of ⁇ -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 ⁇ 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 ⁇ 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.
  • 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).
  • 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 OD 6 oo 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 ⁇ 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.
  • Table I Relative OD max , Ty 2 , V max ( ⁇ 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.
  • 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
  • MDS1 a dosage suppressor of an mckl mutant, encodes a putative yeast homolog of glycogen synthase kinase 3.
  • tau The role of tau (MAPT) in frontotemporal dementia and related tauopathies.

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Abstract

L'invention concerne des cellules de levure qui co-expriment à la fois Αβ et tau et qui présentent une plus grande cytotoxicité que des cellules exprimant tau ou Αβ seul. Ces cellules de levure modélisent les effets synergiques de Αβ et tau, et sont utiles pour identifier des composés qui réduisent la toxicité de Αβ et tau et pour obtenir des espèces tau hyperphosphorylées.
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Citations (2)

* Cited by examiner, † Cited by third party
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WO2002068663A1 (fr) 2001-02-26 2002-09-06 Nv Remynd Modele de tau-opathie
WO2011088059A1 (fr) 2010-01-12 2011-07-21 Whitehead Institute For Biomedical Research Cellules de levure exprimant la bêta amyloïde et leurs utilisations

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Non-Patent Citations (71)

* Cited by examiner, † Cited by third party
Title
ADAMS AE; SHEN W; LIN CS; LEAVITT J; MATSUDAIRA P: "Isoform-specific complementation of the yeast sac6 null mutation by human fimbrin", MOL CELL BIOL, vol. 15, 1995, pages 69 - 75
ALBERTI S; GITLER AD; LINDQUIST S: "A suite of Gateway cloning vectors for high-throughput genetic analysis in Saccharomyces cerevisiae", YEAST, vol. 24, 2007, pages 913 - 919
ALMEIDA CG; TAKAHASHI RH; GOURAS GK: "Beta-amyloid accumulation impairs multivesicular body sorting by inhibiting the ubiquitin-proteasome system", J NEUROSCI, vol. 26, 2006, pages 4277 - 4288
BAGRIANTSEV S; LIEBMAN S: "Modulation of Abeta42 low-n oligomerization using a novel yeast reporter system", BMC BIOL, vol. 4, 2006, pages 32
BARGHORN S; ZHENG-FISCHHOFER Q; ACKMANN M; BIERNAT J; VON BERGEN M; MANDELKOW EM; MANDELKOW E: "Structure, microtubule interactions, and paired helical filament aggregation by tau mutants of frontotemporal dementias", BIOCHEMISTRY, vol. 39, 2000, pages 11714 - 11721
BITTER GA; EGAN KM: "Expression of heterologous genes in Saccharomyces cerevisiae from vectors utilizing the glyceraldehyde-3-phosphate-dehydrogenase gene promoter", GENE, vol. 32, no. 3, 1984, pages 263 - 74
BLURTON-JONES M; LAFERLA FM: "Pathways by which Abeta facilitates tau pathology", CURR ALZHEIMER RES, vol. 3, 2006, pages 437 - 448
BOLAND B; KUMAR A; LEE S; PLATT FM; WEGIEL J; YU WH; NIXON RA: "Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease", J NEUROSCI, vol. 28, 2008, pages 6926 - 6937
BUEE-SCHERRER V; CONDAMINES O; MOURTON-GILLES C; JAKES R; GOEDERT M; PAU B; DELACOURTE A: "AD2, a phosphorylation-dependent monoclonal antibody directed against tau proteins found in Alzheimer's disease", BRAIN RES MOL BRAIN RES, vol. 39, 1996, pages 79 - 88
CARMONA-GUTIERREZ D; EISENBERG T; BUTTNER S; MEISINGER C; KROEMER G; MADEO F: "Apoptosis in yeast: triggers, pathways, subroutines", CELL DEATH DIFFER, vol. 17, 2010, pages 763 - 773
CASPERSEN C; WANG N; YAO J; SOSUNOV A; CHEN X; LUSTBADERJW; XU HW; STERN D; MCKHANN G; YAN SD: "Mitochondrial Abeta: a potential focal point for neuronal metabolic dysfunction in Alzheimer's. disease", FASEB J, vol. 19, 2005, pages 2040 - 2041
D'ANGELO F; VIGNAUD H; DI MARTINO J; SALIN B; DEVIN A; CULLIN C; MARCHAL C: "A yeast model for amyloid beta aggregation exemplifies the role of membrane trafficking and PICALM in cytotoxicity", DIS MODEL MECH, vol. 6, 2013, pages 206 - 216
DAROCHA-SOUTO B; COMA M; PEREZ NIEVAS BG; SCOTTON TC; SIAO M; SANCHEZ-FERRER P; HASHIMOTO T; FAN Z; HUDRY E; BARROETA I: "Activation of glycogen synthase kinase-3 beta mediates beta-amyloid induced neuritic damage in Alzheimer's disease", NEUROBIOL DIS, vol. 45, 2012, pages 425 - 437
EDBAUER D; WINKLER E; REGULA JT; PESOLD .B; STEINER H; HAASS C: "Reconstitution of gamma-secretase activity", NAT CELL BIOL, vol. 5, 2003, pages 486 - 488
FITZGERALD; GLICK: "Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targeting", MICROB CELL FACTORIES, vol. 13, no. 1, 2014, pages 125
FOLWELL J; COWAN CM; UBHI KK; SHIABH H; NEWMAN TA; SHEPHERD D; MUDHER A: "Abeta exacerbates the neuronal dysfunction caused by human tau expression in a Drosophila model of Alzheimer's disease", EXP NEUROL, vol. 223, 2010, pages 401 - 409
FRANSSENS V; BYNENS T; VAN DEN BRANDE J; VANDERMEEREN K; VERDUYCKT M; WINDERICKX J: "The benefits of humanized yeast models to study Parkinson's disease", OXID MED CELL LONGEV, 2013, pages 760629
GIETZ D; ST JEAN A; WOODS RA; SCHIESTL RH: "Improved method for high efficiency transformation of intact yeast cells", NUCLEIC ACIDS RES, vol. 20, 1992, pages 1425
GINSBERG SD; ALLDRED MJ; COUNTS SE; CATALDO AM; NEVE RL; JIANG Y; WUU J; CHAO MV; MUFSON EJ; NIXON RA: "Microarray analysis of hippocampal CA1 neurons implicates early. endosomal dysfunction during Alzheimer's disease progression", BIOL PSYCHIATRY, vol. 68, 2010, pages 885 - 893
GLENNER GG; WONG CW: "Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein", BIOCHEM BIOPHYS RES COMMUN, vol. 120, 1984, pages 885 - 890
GOEDERT M; WISCHIK CM; CROWTHER RA; WALKER JE; KLUG A: "Cloning and sequencing 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, vol. 85, 1988, pages 4051 - 4055
GOTZ J; DETERS N; DOLDISSEN A; BOKHARI L; KE Y; WIESNER A; SCHONROCK N; ITTNER LM: "A decade of tau transgenic animal models and beyond", BRAIN PATHOL, vol. 17, 2007, pages 91 - 103
GRUNDKE-LQBAL I; IQBAL K; TUNG YC; QUINLAN M; WISNIEWSKI HM; BINDER LI: "Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology", PROC NATL ACAD SCI U S A, vol. 83, 1986, pages 4913 - 4917
HAGIWARA M; SHINOMIYA H; KASHIHARA M; KOBAYASHI K; TADOKORO T; YAMAMOTO Y: "Interaction of activated Rab5 with actin-bundling proteins, L- and T-plastin and its relevance to endocytic functions in mammalian cells", BIOCHEM BIOPHYS RES COMMUN, vol. 407, 2011, pages 615 - 619
HARDY J; SELKOE DJ: "The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics", SCIENCE, vol. 297, 2002, pages 353 - 356
HARDY TA; WU D; ROACH PJ: "Novel 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, vol. 208, 1995, pages 728 - 734
HAROLD D; ABRAHAM R; HOLLINGWORTH P; SIMS R; GERRISH A; HAMSHERE ML; PAHWA JS; MOSKVINA V; DOWZELL K; WILLIAMS A: "Genome-wide association study identifies variants at CLU and PICALM associated with Alzheimer's disease", NAT GENET, vol. 41, 2009, pages 1088 - 1093
HERNANDEZ F; GOMEZ DE BARREDA E; FUSTER-MATANZO A; LUCAS JJ; AVILA J: "GSK3: a possible link between beta amyloid peptide and tau protein", EXP NEUROL, vol. 223, 2010, pages 322 - 325
HERNANDEZ F; LUCAS JJ; AVILA J: "GSK3 and tau: two convergence points in Alzheimer's disease", J ALZHEIMERS DIS, vol. 33, no. 1, 2013, pages 141 - 144
ITTNER LM; GOTZ J: "Amyloid-beta and tau--a toxic pas de deux in Alzheimer's disease.", NAT REV NEUROSCI, vol. 12, 2011, pages 65 - 72
ITTNER LM; KE YD; DELERUE F; BI M; GLADBACH A; VAN EERSEL J; WOLFING H; CHIENG BC; CHRISTIE MJ; NAPIER IA: "Dendritic function of tau mediates amyloid-beta toxicity in Alzheimer's disease mouse models", CELL, vol. 142, 2010, pages 387 - 397
JOHNSTON M; DAVIS RW: "Sequences that regulate the divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae", MOL. CELL. BIOL., vol. 4, no. 8, 1984, pages 1440
KEIL U; HAUPTMANN S; BONERT A; SCHERPING I; ECKERT A; MULLER WE: "Mitochondrial dysfunction induced by disease relevant AbetaPP and tau protein mutations", J ALZHEIMERS DIS, vol. 9, 2006, pages 139 - 146
LEWIS J; DICKSON DW; LIN WL; CHISHOLM L; CORRAL A; JONES G; YEN SH; SAHARA N; SKIPPER L; YAGER D: "Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP", SCIENCE, vol. 293, 2001, pages 1487 - 1491
LLORENS-MARTIN M; JURADO J; HERNANDEZ F; AVILA J: "GSK-3beta, a pivotal kinase in Alzheimer disease", FRONT MOL NEUROSCI, vol. 7, 2014, pages 46
LOMBARDI R; RIEZMAN H: "Rvs161p and Rvsl67p, the two yeast amphiphysin homologs, function together in vivo", J BIOL CHEM, vol. 276, 2001, pages 6016 - 6022
LUTHI U; SCHAERER-BRODBECK C; TANNER S; MIDDENDORP 0; EDLER K; BARBERIS A: "Human beta-secretase activity in yeast detected by a novel cellular growth selection system", BIOCHIM BIOPHYS ACTA, vol. 1620, 2003, pages 167 - 178
MAGER WH; WINDERICKX J: "Yeast as a model for medical and medicinal research", TRENDS PHARMACOL SCI, vol. 26, 2005, pages 265 - 273
MANCZAK M; ANEKONDA TS; HENSON E; PARK BS; QUINN J; REDDY PH: "Mitochondria 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, vol. 15, 2006, pages 1437 1449
MASTERS CL; SIMMS G; WEINMAN NA; MULTHAUP G; MCDONALD BL; BEYREUTHER K: "Amyloid plaque core protein in Alzheimer disease and Down syndrome", PROC NATL ACAD SCI USA, vol. 82, 1985, pages 4245 - 4249
MOUSSA CE; FU Q; KUMAR P; SHTIFMAN A; LOPEZ JR; ALLEN PD; LAFERLA F; WEINBERG D; MAGRANE J; APRAHAMIAN T: "Transgenic expression of beta-APP in fast-twitch skeletal muscle leads to calcium dyshomeostasis and IBM-like pathology", FASEBJ, vol. 20, 2006, pages 2165 - 2167
ODDO S; CACCAMO A; SHEPHERD JD; MURPHY MP; GOLDE TE; KAYED R; METHERATE R; MATTSON MP; AKBARI Y; LAFERLA FM: "Triple-transgenic model of Alzheimer's disease with plaques and tangles: intracellular Abeta and synaptic dysfunction", NEURON, vol. 39, 2003, pages 409 - 421
PARK H; KAM TL; KIM Y; CHOI H; GWON Y; KIM C; KOH JY; JUNG YK: "Neuropathogenic role of adenylate kinase-1 in Abeta-mediated tau phosphorylation via AMPK and GSK3beta", HUM MOL GENET, vol. 21, 2012, pages 2725 - 2737
PICARD D; KHURSHEED B; GARABEDIAN MJ; FORTIN MG; LINDQUIST S; YAMAMOTO KR: "Reduced levels of hsp90 compromise steroid receptor action in vivo", NATURE, vol. 348, no. 6297, 8 December 1989 (1989-12-08), pages 166 - 8
PORZIG R; SINGER D; HOFFMANN R: "Epitope mapping of mAbs AT8 and Tau5 directed against hyperphosphorylated regions of the human tau protein", BIOCHEM BIOPHYS RES COMMUN, vol. 358, 2007, pages 644 - 649
PRÜ?ING K; VOIGT A; SCHULZ JB: "Drosophila melanogaster as a model organism for Alzheimer's disease", MOL NEURODEGENER, vol. 8, 2013, pages 35
PUZISS JW; HARDY TA; JOHNSON RB; ROACH PJ; HIETER P: "MDS1, a dosage suppressor of an mckl mutant, encodes a putative yeast homolog of glycogen synthase kinase 3", MOL CELL BIOL, vol. 14, 1994, pages 831 - 839
RADEMAKERS R; CRUTS M; VAN BROECKHOVEN C: "The role of tau (MAPT) in frontotemporal dementia and related tauopathies", HUM MUTAT, vol. 24, 2004, pages 277 - 295
RAJENDRAN L; ANNAERT W: "Membrane trafficking pathways in Alzheimer's disease", TRAFFIC, vol. 13, 2012, pages 759 - 770
RAPOPORT M; DAWSON HN; BINDER LI; VITEK MP; FERREIRA A: "Tau is essential to beta -amyloid-induced neurotoxicity", PROC NATL ACAD SCI U S A, vol. 99, 2002, pages 6364 - 6369
RHEIN V; SONG X; WIESNER A; ITTNER LM; BAYSANG G; MEIER F; OZMEN L; BLUETHMANN H; DROSE S; BRANDT U: "Amyloid-beta and tau synergistically impair the oxidative phosphorylation system in triple transgenic Alzheimer's disease mice", PROC NATL ACAD SCI US A, vol. 106, 2009, pages 20057 - 20062
SERENO , L; COMA M; RODRIGUEZ M; SANCHEZ-FERRER P; SANCHEZ MB; GICH I; AGULLO JM; PEREZ M; AVILA J; GUARDIA-LAGUARTA C: "A novel GSK-3beta inhibitor reduces Alzheimer's pathology and rescues neuronal loss in vivo", NEUROBIOL D/S, vol. 35, 2009, pages 359 - 367
SERGEANT N; DELATOURTE A; BUEE L: "Tau protein as a -differential biomarker of tauopathies", BIOCHIM BIOPHYS ACTA, vol. 1739, 2005, pages 179 - 197
SOFOLA 0; KERR F; ROGERS I; KILLICK R; AUGUSTIN H; GANDY C; ALLEN MJ; HARDY J; LOVESTONE S; PARTRIDGE L: "Inhibition of GSK-3 ameliorates Abeta pathology in an adult-onset Drosophila model of Alzheimer's disease", PLOS GENET, vol. 6, 2010, pages E1001087
TERWEL D; MUYLLAERT D; DEWACHTER I; BORGHGRAEF P; CROES S; DEVIJVER H; VAN LEUVEN F: "Amyloid activates GSK-3beta to aggravate neuronal tauopathy in bigenic mice", AM J PATHOL, vol. 172, 2008, pages 786 - 798
TOWBIN H; STAEHELIN T; GORDON J: "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications", PROC NATL AC?D SCI USA, vol. 76, 1979, pages 4350 - 4354
TREUSCH S; HAMAMICHI S; GOODMAN JL; MATLACK KE; CHUNG CY; BARU V; SHULMAN JM; PARRADO A; BEVIS BJ; VALASTYAN JS: "Functional links between Abeta toxicity, endocytic trafficking, and Alzheimer's disease risk factors in yeast", SCIENCE, vol. 334, 2011, pages 1241 - 1245
UMEDA T; MAEKAWA S; KIMURA T; TAKASHIMA A; TOMIYAMA T; MORI H: "Neurofibrillary tangle formation by introducing wild-type human tau into APP transgenic mice", ACTA NEUROPATHOL, vol. 127, 2014, pages 685 - 698
VAN SWIETEN JC; BRONNER IF; AZMANI A; SEVERIJNEN LA; KAMPHORST W; RAVID R; RIZZU P; WILLEMSEN R; HEUTINK P: "The DeltaK280 mutation in MAP tau favors exon 10 skipping in vivo", J NEUROPATHOL EXP NEUROL, vol. 66, 2007, pages 17 - 25
VANDEBROEK T; TERWEL D; VANHELMONT T; GYSEMANS M; VAN HAESENDONCK C; ENGELBORGHS Y; WINDERICKX J; VAN LEUVEN F: "Microtubule binding and clustering of human Tau-4R and Tau-P301L proteins isolated from yeast deficient in orthologues of glycogen synthase kinase-3beta or cdk5", J BIOL CHEM, vol. 281, 2006, pages 25388 - 25397
VANDEBROEK T; VANHELMONT T; TERWEL D; BORGHGRAEF P; LEMAIRE K; SNAUWAERT J; WERA S; VAN LEUVEN F; WINDERICKX J: "Identification and isolation of a hyperphosphorylated, conformationally changed intermediate of human protein tau expressed in yeast", BIOCHEMISTRY, vol. 44, 2005, pages 11466 - 11475
VANHELMONT T; VANDEBROEK T; DE VOS A; TERWEL D; LEMAIRE K; ANANDHAKUMAR J; FRANSSENS V; SWINNEN E; VAN LEUVEN F; WINDERICKX J: "Serine-409 phosphorylation and oxidative damage define aggregation of human protein tau in yeast", FEMS YEAST RES, vol. 10, 2010, pages 992 - 1005
VIGNAUD H; BOBO C; LASCU I; SORGJERD KM; ZAKO T; MAEDA M; SALIN B; LECOMTE S; CULLIN C: "A structure-toxicity study of Ass42 reveals a new anti-parallel aggregation pathway", PLOS ONE, vol. 8, 2013, pages E80262
VON BERGEN M; BARGHORN S; LI L; MARX A; BIERNAT J; MANDELKOW EM; MANDELKOW E: "Mutations of - tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure", J BIOL CHEM, vol. 276, 2001, pages 48165 - 48174
WACH A; BRACHAT A; POHLMANN R; PHILIPPSEN P: "New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae", YEAST, vol. 10, 1994, pages 1793 - 1808
WALDO GS; STANDISH BM; BERENDZEN J; TERWILLIGER TC: "Rapid ,protein-folding assay using green_ fluorescent protein", NATURE BIOTECHNOL., vol. 17, 1990, pages 691 - 695
WHITEMAN IT; MINAMIDE LS; GOH DE L; BAMBURG JR; GOLDSBURY C: "Rapid changes in phospho-MAP/tau epitopes during neuronal stress: cofilin-actin rods primarily recruit microtubule binding domain epitopes", PLOS ONE, vol. 6, 2011, pages E20878
WINDERICKX J; DELAY C; DE VOS A; KLINGER H; PELLENS K; VANHELMONT T; VAN LEUVEN F; ZABROCKI P: "Protein folding diseases arid neurodegeneration: lessons learned from yeast", BIOCHIM BIOPHYS ACTA, vol. 1783, 2008, pages 1381 - 1395
WITTMANN CW; WSZOLEK MF; SHULMAN JM; SALVATERRA PM; LEWIS J; HUTTON M; FEANY MB: "Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles", SCIENCE, vol. 293, 2001, pages 711 - 714
ZHANG H; KOMANO H; FULLER RS; GANDY SE; FRAIL DE: "Proteolytic processing and secretion of human beta-amyloid :precursor protein in yeast. Evidence for a yeast secretase activity", J BIOL CHEM, vol. 269, 1994, pages 27799 - 27802
ZHANG W; ESPINOZA D; HINES V; INNIS M; MEHTA P; MILLER DL: "Characterization of beta-amyloid peptide precursor processing by the yeast Yap3 and Mkc7 proteases", BIOCHIM BIOPHYS ACTA, vol. 1359, 1997, pages 110 - 122

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