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Review
. 2011 Mar;53(3):238-45.
doi: 10.1016/j.ymeth.2010.11.006. Epub 2010 Nov 27.

TDP-43 toxicity in yeast

Maria Armakola  1 , Michael P Hart, Aaron D Gitler
Affiliations
Review

TDP-43 toxicity in yeast

Maria Armakola et al. Methods. 2011 Mar.

Abstract

The budding yeast Saccharomyces cerevisiae is an emerging tool for investigating the molecular pathways that underpin several human neurodegenerative disorders associated with protein misfolding. Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disease primarily affecting motor neurons. The protein TDP-43 has recently been demonstrated to play an important role in the disease, however, the mechanisms by which TDP-43 contributes to pathogenesis are unclear. To explore the mechanistic details that result in aberrant accumulation of TDP-43 and to discover potential strategies for therapeutic intervention, we employed a yeast TDP-43 proteinopathy model system. These studies allowed us to determine the regions of TDP-43 required for aggregation and toxicity and to define the effects of ALS-linked mutant forms of TDP-43. We have also been able to harness the power of yeast genetics to identify potent modifiers of TDP-43 toxicity using high-throughput yeast genetic screens. Here, we describe the methods and approaches that we have used in order to gain insight into TDP-43 biology and its role in disease. These approaches are readily adaptable to other neurodegenerative disease proteins.

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Figures

Figure 1
Figure 1
Yeast TDP-43 proteinopathy model. A TDP-43 expression construct under the control of a tightly regulated galactose-inducible promoter was introduced into yeast cells. A) Spotting assays using five-fold serial dilutions demonstrates TDP-43 is toxic when expressed at high levels in yeast cells (pAG426Gal-TDP-43-GFP). When cells are spotted on plates containing glucose, TDP-43 expression is repressed and induced in the presence of galactose. B) TDP-43-GFP formed multiple cytoplasmic foci when expressed in yeast cells, whereas GFP alone was diffusely distributed throughout the cytoplasm and nucleus.
Figure 2
Figure 2
Accelerated aggregation by mutant TDP-43 linked to ALS. A) Yeast cells expressing YFP-tagged WT or Q331K TDP-43 at low levels. WT cells rarely contain > 4 foci per cell, whereas Q331K cells contain many more aggregates. B) Quantification of increase in aggregation by Q331K performed in triplicate by researcher blinded to identities of samples. C) Spotting assays demonstrate Q331K mutant TDP-43 is much more toxic than WT, despite being expressed at equivalent levels (data not shown).
Figure 3
Figure 3
Yeast strains engineered to express various levels of TDP-43. Spotting assays demonstrate galactose-inducible expression of YFP alone, or low, intermediate, and high levels of TDP-43 expression, which result in a corresponding level of toxicity.
Figure 4
Figure 4
Yeast plasmid overexpression screen to identify modifiers of TDP-43 toxicity. A) Schematic of yeast genetic screen. Yeast cells harboring an integrated galactose-inducible TDP-43 cassette, were individually transformed with a library of 5,500 yeast open reading frames (ORFs) and spotted onto galactose plates to induce expression of TDP-43 and each gene from the library. B) A representative plate from the yeast screen. Each spot represents a yeast strain expressing TDP-43 along with one gene from the library. Examples of genes that suppressed TDP-43 toxicity (improved growth) are indicated by green arrows and enhancers of toxicity (inhibited growth) are indicated by red arrows.
Figure 5
Figure 5
Yeast deletion screen to identify modifiers of TDP-43 toxicity. A) Schematic of yeast deletion screen, based on (51). The galactose-inducible TDP-43 expression construct (pAG416Gal-TDP-43) was introduced into MATα strain Y7092 to generate the query strain. This query strain was mated to the yeast haploid deletion collection of non-essential genes (MATa, each gene deleted with KanMX cassette (confers resistance to G418). Mating, sporulation, and mutant selection were performed using a Singer RoToR HDA (Singer Instruments, Somerset, UK). Haploid mutants harboring the TDP-43 expression plasmid were grown in the presence of glucose (TDP-43 expression “off”) or galactose (TDP-43 expression “on”). Following growth at 30 °C for 2 days, plates were photographed and colony sizes measured by ImageJ image analysis software, based on (54). B) A representative plate from the deletion screen. Left is glucose (deletion alone, e.g. xxxΔ) and right is galactose (deletion + TDP-43 expression, e.g. xxxΔ + TDP-43). Each plate contains 384 different strains pinned in duplicate (768 total). The red arrows point to a strong alleviating genetic interaction (toxicity suppressor), in which the gene deletion + TDP-43, grows better than TDP-43- or the deletion alone.
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