Aggregate-like perimitochondrial foci upon expression of TDP-43 in yeast. YFP-tagged human wild-type TDP-43 and ALS-associated TDP-43-Q331K were co-expressed overnight with red fluorescent mtmCherry from a low copy (CEN6) and a high copy (2μ) yeast expression plasmid, respectively, both under the control of a GAL1 promoter in growth medium with galactose as the sole carbon source. Before microscopy, cultures were diluted in expression medium and incubated for 4 h to reach logarithmic growth phases. Images represent single layers applying confocal microscopy. Scale bars, 2.5 μm. Pseudocolor images are shown. ctrl, control; BF, bright field.

Apoptotic and necrotic cell death in TDP-43-expressing yeast cells. TDP-43 was expressed from the low copy (CEN6) expression plasmid as in Fig. 2. Two days after inducing TDP-43 expression, yeast cells were evaluated for morphological markers of cell death. Annexin V (A)⁺/PI⁻ and Annexin V⁻/PI⁺ yeast cells are referred to as (early) apoptotic and necrotic, respectively. Annexin V⁺/PI⁺ yeast cells are referred to as late apoptotic and secondary necrotic. A, quantitative measurement of cell death using FACS analysis. Data represent mean values of five independent experiments. Error bars represent S.E. p values were determined with a t test by comparing vector controls with TDP-43-WT and TDP-43-Q331K, respectively. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. B, Annexin V/PI-stained cells from A analyzed by fluorescence microscopy. Scale bar, 5 μm. Pseudocolor images are shown. ctrl, control; DIC, differential interference contrast.

Depleting respiratory chain complexes relieves TDP-43-triggered cytotoxicity. TDP-43 was expressed from a high copy (2μ) yeast expression plasmid under the control of a GAL10 promoter in liquid nutrient-containing growth medium with glucose and galactose as in Fig. 4. Panel 1, TDP-43 was expressed in wild-type and knock-out strains for 14 h and evaluated for effects on clonogenicity in knock-out strains deleted for integral membrane proteins of the respiratory chain complexes III–V (A–C). The mean values of cfus obtained using yeast cells expressing vector controls were set to 100%. Data represent mean values of at least three independent experiments. Error bars represent S.E. p values were determined with a t test by comparing relative clonogenicity of TDP-43-WT in the wild-type strain versus knock-out strains. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Panel 2, respiratory capacity of the indicated knock-out strains using a clonogenic approach to measure the growth of colonies on solid nutrient-containing growth medium with glycerol as the sole (respiratory) carbon source. The clonogenic respiratory capacity of the wild-type strain was set to 100%. Data represent mean values of three independent experiments except those involving Δatp18 for which two experiments were performed. Error bars represent S.E. p values were determined with a t test by comparing clonogenic respiratory capacities of knock-out strains with those of the wild-type strain. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Panel 3, correlation of clonogenicity upon TDP-43 expression (see panel 1) with clonogenic respiratory capacity of the respective yeast strains (see panel 2). The coefficient of determination, R², was determined using linear regression (Microsoft Excel 2010). Panel 4, mtDNA content in the indicated knock-out strains. The proportion of cells lacking extranuclear mtDNA nucleoids was determined by DAPI staining. Data represent mean values of at least four independent experiments. Error bars represent S.E. p values were determined with a t test by comparing knock-out strains with the wild-type strain. *, p < 0.05; **, p < 0.01. Panel 5, correlation of clonogenicity upon TDP-43 expression (see panel 1) with mtDNA content of the respective yeast strains (see panel 4). The coefficient of determination, R², was determined using logarithmic regression (Microsoft Excel 2010). ctrl, control.

Increased respiratory capacity exacerbates TDP-43-triggered cytotoxicity and cell death. A–D, yeast cells transformed with a high copy (2μ) yeast expression plasmid under the control of a GAL10 promoter were precultured in either fermentative (glucose) or obligatory respiratory media (glycerol) for 2 days. TDP-43 expression was induced by shifting cells to liquid growth medium containing both glucose and galactose as in Fig. 4. A, yeast cells were evaluated for clonogenicity. The mean value of the cfus obtained using yeast cells expressing vector controls was set to 100%. Data represent mean values of at least four independent experiments. Error bars represent S.E. *, p < 0.05; **, p < 0.01 (t test). B and C, oxidative stress levels (DHE staining) and incidences of membrane permeabilization (PI staining) were measured by a fluorescence plate reader at day 2 after inducing TDP-43 expression. Data represent mean values of five independent experiments. Error bars represent standard error. *, p < 0.05; **, p < 0.01 (t test, paired). D, at 2 days of TDP-43 expression, yeast cells were evaluated quantitatively using FACS analysis to detect morphological markers of cell death. Annexin V (A)⁺/PI⁻ and Annexin V⁻/PI⁺ yeast cells are referred to as (early) apoptotic and necrotic cells, respectively, whereas Annexin V⁺/PI⁺ yeast cells are referred to as late apoptotic and secondary necrotic cells. Data represent mean values of three experiments performed in parallel. Error bars represent S.E. **, p < 0.01 (t test). E, yeast cells were co-transformed with a high copy (2μ) yeast expression plasmid encoding TDP-43 under the control of a GAL10 promoter and with a low copy (CEN4) yeast expression plasmid encoding NDI1 under the control of a GAL1 promoter. TDP-43 and NDI1 expression was induced by shifting cells to liquid growth medium containing both glucose and galactose as in Fig. 4. Oxidative stress levels (DHE staining) were measured by a fluorescence plate reader 2 days after inducing TDP-43 and NDI1 expression. Data represent mean values of four independent experiments. Error bars represent S.E. *, p < 0.05 (t test). ctrl, control; RFU, relative fluorescent units.

Age-dependent TDP-43-triggered cytotoxicity in yeast. Wild-type TDP-43 and TDP-43-Q331K (both untagged) were expressed from a low copy (CEN6) yeast expression plasmid under the control of a GAL1 promoter in growth medium with galactose as the sole carbon source. A, immunoblot confirming TDP-43 expression. Rpl16p was used as loading control. B, yeast cells were evaluated for clonogenicity at the indicated time points after induction of expression. Data represent mean values of at least five independent experiments. Error bars represent S.E. C and D, oxidative stress levels (DHE staining) and incidences of membrane permeabilization (PI staining) were measured using a fluorescence plate reader at the indicated time points after induction of TDP-43 expression. Data represent mean values of at least three independent experiments. Error bars represent S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (t test, paired). E and F, DHE- and PI-stained cells from C and D collected at day 2 after induction of TDP-43 expression were analyzed by fluorescence microscopy. Please note that plasma membrane permeabilization upon TDP-43 expression was already reported (13). Scale bars, 5 μm. Pseudocolor images are shown. ctrl, control; RFU, relative fluorescence units; d, day(s).

Depletion from mtDNA relieves TDP-43-triggered cytotoxicity. A, TDP-43 was expressed from a high copy (2μ) yeast expression plasmid under the control of a GAL10 promoter in wild-type ρ⁺ and mtDNA-depleted ρ⁰ strains in nutrient-containing growth medium with glucose for fermentative growth and galactose for the induction of TDP-43 expression. Yeast cultures were evaluated for clonogenicity at the indicated time points. Data represent mean values of at least three independent experiments. Error bars represent S.E. B, TDP-43 was expressed as described in A in wild-type and knock-out strains deleted for genes encoding yeast cell death proteases for 14 h and evaluated for clonogenicity. The mean value of the cfus of yeast cells expressing vector controls was set to 100%. Data represent mean values of at least four independent experiments. Error bars represent S.E. p values were determined with a t test by comparing relative clonogenicity of TDP-43-WT in the wild-type strain versus knock-out strains. *, p < 0.05. ctrl, control; Glc, glucose; d, day(s).

TDP-43-triggered cytotoxicity is not exacerbated by pharmacologically inhibiting respiratory chain complexes. TDP-43 was expressed from a high copy (2μ) yeast expression plasmid under the control of a GAL10 promoter in liquid growth medium with glucose and galactose as in Fig. 4. TDP-43 was expressed in the wild-type strain for 15 h in the presence of the respiratory chain inhibitors antimycin A (10 μm) (A), myxothiazol (1 μm) (B), and oligomycin (1 μg/ml) (C) and evaluated for clonogenicity. The mean values of the cfus obtained using untreated yeast cells expressing vector controls were set to 100% in each experiment. Data represent mean values of six independent experiments. Error bars represent S.E. *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 (t test). ctrl, control.