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Comparative Study
. 2011 Jun 15;20(12):2308-21.
doi: 10.1093/hmg/ddr124. Epub 2011 Mar 26.

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS

Patricia S Estes  1 , Ashley Boehringer, Rebecca Zwick, Jonathan E Tang, Brianna Grigsby, Daniela C Zarnescu
Affiliations
Comparative Study

Wild-type and A315T mutant TDP-43 exert differential neurotoxicity in a Drosophila model of ALS

Patricia S Estes et al. Hum Mol Genet. .

Abstract

The RNA-binding protein TDP-43 has been linked to amyotrophic lateral sclerosis (ALS) both as a causative locus and as a marker of pathology. With several missense mutations being identified within TDP-43, efforts have been directed towards generating animal models of ALS in mouse, zebrafish, Drosophila and worms. Previous loss of function and overexpression studies have shown that alterations in TDP-43 dosage recapitulate hallmark features of ALS pathology, including neuronal loss and locomotor dysfunction. Here we report a direct in vivo comparison between wild-type and A315T mutant TDP-43 overexpression in Drosophila neurons. We found that when expressed at comparable levels, wild-type TDP-43 exerts more severe effects on neuromuscular junction architecture, viability and motor neuron loss compared with the A315T allele. A subset of these differences can be compensated by higher levels of A315T expression, indicating a direct correlation between dosage and neurotoxic phenotypes. Interestingly, larval locomotion is the sole parameter that is more affected by the A315T allele than wild-type TDP-43. RNA interference and genetic interaction experiments indicate that TDP-43 overexpression mimics a loss-of-function phenotype and suggest a dominant-negative effect. Furthermore, we show that neuronal apoptosis does not require the cytoplasmic localization of TDP-43 and that its neurotoxicity is modulated by the proteasome, the HSP70 chaperone and the apoptosis pathway. Taken together, our findings provide novel insights into the phenotypic consequences of the A315T TDP-43 missense mutation and suggest that studies of individual mutations are critical for elucidating the molecular mechanisms of ALS and related neurodegenerative disorders.

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Figures

Figure 1.
Figure 1.
Overexpression of human wild-type and A315T mutant TDP-43 leads to neurodegeneration in the adult retina. (A–F) Expression of hwt and hA315T results in normal surface phenotypes at 25°C (B and C compared with A). Both hTDP-43 transgenes exhibit strong surface phenotypes when expressed at higher levels (29°C, see E and F) compared with controls (D). Genotypes as indicated. Anterior right, dorsal up. (A′–F′) Corresponding plastic sections indicate that regardless of the surface phenotype, the retinas undergo cell loss. Note large areas of cell loss within the retina (arrows). Adult eyes shown are from 1–2-day-old flies. (G) Western blot analyses showing hTDP-43 expression. Note similar levels of TDP-43 expression for the wild-type (hwt) and A315T (hA315T) transgenes as well as increased expression levels in an additional hA315T line (hA315T HE). Genotypes as indicated, on top. Blotting antibodies as indicated on the right. Tubulin was used as a loading control. (H) Quantification of relative protein levels from Western blot analysis. Scale bar (A′): 30 µm.
Figure 2.
Figure 2.
Overexpression of TDP-43 in larval eye imaginal discs leads to altered cytoplasmic localization and axonal aggregates. (A–F) Single-confocal slices (1 µm each) showing hTDP-43 localization when expressed in the developing retina with GMR-Gal4. hTDP-43 visualized via individual fluorescent tags [as indicated (B, C and E–F″), compare with GFP-NLS (A, D–D″)]. Filamentous actin labeled with phalloidin (phall), and DNA stained with Hoechst (as indicated). Note TDP-43 aggregates in axons (arrowheads). (D′–F′) High magnification views of optic stalk show TDP-43 aggregates in axons (white arrowheads, D′–F′), whereas GFP-NLS remains restricted to nuclei (D′). (D″–F″) High-magnification insets showing the localization of GFP-NLS (D″) and TDP-43 wt and A315T in relation to the nucleus (E″–F″). Stainings as indicated. Note that hwt forms more pronounced aggregates than hA315T, which appears more diffusely distributed (asterisk). Individual nuclei are circled in red. Red arrowheads indicate some amount of depletion from the nucleus hwt. Scale bar (A): 50 µm.
Figure 3.
Figure 3.
Subcellular localization of TDP-43 in motor neurons. (A–E) D42-Gal4-driven expression of GFP-NLS (A, control) and TDP-43 variants (B–E) in ventral ganglia of third instar larvae. Genotypes indicated on the top, and stainings shown on the left. Images shown represent projections of 1 µm confocal slices. GFP (or YFP) and RFP indicate tags used to visualize TDP-43 variants, and DNA visualized using Hoechst. (A′–E″) High-magnification views of ventral ganglia shown in (A)–(E). Both human transgenes (wt and A315T) remained restricted to the nucleus (white arrows in B and B′, and C and C′). Note cell with peripheral nuclear localization in hTDP-43 wt (white arrowhead in B and B′). Wild-type TBPH translocates to the cytoplasm and forms axonal aggregates (red arrows in D′). The fly A315T mutant protein is mostly restricted to the nucleus (white arrows in E and E′). Hoechst staining of the samples shown in (A′)–(E′) labels DNA. Scale bars: 30 μm in (A), 15 μm in (A′).
Figure 4.
Figure 4.
NMJ morphology is altered by overexpression of TDP-43 variants. (A–H″) Larval NMJs at muscles 6/7, abdominal segment A3 (A–D″) and abdominal segment A6 (E–H″). Genotypes shown on the left, and stainings indicated on the top. Selected terminal type 1b boutons (marked with asterisks) are shown in (A′–D′) and (A′–D′, HRP only), and likewise for A6 in (E′–H′) and (E′–H′, HRP only). Arrowheads indicate thinning of the HRP-stained neuronal membrane. Arrows indicate satellite boutons. D42-Gal4-driven overexpression of TDP-43 (wild-type and A315T) affects various aspects of synaptic morphology [see (I) for quantitative analyses at A3, which include a high expressing hA315T transgene (A315T HE)]. Student's t-test was used to determine statistical significance. ***P< 0.001; **P< 0.01; *P< 0.05. Scale bar (A): 45 μm.
Figure 5.
Figure 5.
Overexpression of TDP-43 variants acts as a dominant negative and affects locomotor activity and survival. (A) Larvae expressing D42-driven human wild-type, hA315T mutant TDP-43 (including hA315T HE) and TBPH RNAi transgenes (alone or in combination) take significantly longer to turn over following a ventral-up inversion. Genotypes as indicated. (B) hTDP-43 expression or TBPH RNAi in motor neurons cause a dramatic decrease in adult survival. (C and D) Adult climbing assays performed on the adult survivors show severe motor impairment at both 18 (C) and 25°C (D). Student's t-test was used to determine statistical significance. ***P< 0.001; **P< 0.01; *P< 0.05.
Figure 6.
Figure 6.
Motor neuron apoptosis due to TDP-43 overexpression. (A–D) TUNEL staining marks apoptotic cells. Genotypes/treatment indicated on top, and stainings shown on the left. (A and A′) RFP-NLS expressing ventral ganglia treated with HCl exhibits widespread TUNEL staining. (B and B′) RFP-NLS expressing ventral ganglia shows no indication of cell death (negative control). (C and C′) Wild-type TBPH expression results in some apoptotic cells within the ventral ganglia (arrows, compare C′ with A′ and B′). (D and D′) Few apoptotic cells can be detected in A315T TBPH expressing ventral ganglia (arrows). (E) GFP NLS expression in adult motor neurons using D42 Gal4. Thoracic segments T1, 2, 3 as shown. Arrows (E–G) indicate areas where motor neurons are located. Note: The region marked by red inset in (E) is shown at high magnification for different thoracic ganglia (assayed for apoptosis) in (E1′)–(G″). (E1′ and E1″) TUNEL staining in the T1/T2 region of a GFP-NLS adult thoracic ganglia treated with HCl (positive control). (E2′ and E2″) GFP-NLS expression does not induce apoptosis (negative control). (F′–G″) Adult thoracic ganglia expressing hwt (F′ and F″) and hA315T mutant (G′ and G″). Motor neurons visualized via fluorescent protein tag when either hwt (F) or hA315T mutant TDP-43 (G) is expressed with the D42 driver. Note the dramatic reduction in motor neurons as a result of hwt expression (F). TUNEL assays indicate the presence of apoptotic cells (arrows) when hwt (F′ and F″) or, to a lesser extent, A315T mutant hTDP-43 (G′ and G″) are expressed in adult motor neurons. Scale bar: 70 μm (A), 125 μm (E) and 70 μm (E1′).
Figure 7.
Figure 7.
TDP-43 toxicity is modulated by the proteasome, HSP70 activities and the apoptosis pathway. (A and E) Compound eyes expressing hwt (A) or A315T mutant hTDP-43 (hA315T, E) at 25°C, in 15-day-old adults, exhibit age-dependent depigmentation. Genotypes as indicated on the left, and interacting genes on the top. All transgenes expressed with GMR-Gal4. (B and F) Coexpression of prosβ enhances the depigmentation phenotype due to TDP-43 overexpression (arrows). (C, D, G and H) Coexpression of Hsp70 (C and G) or the p35 caspase inhibitor (D and H) alleviates the eye depigmentation phenotypes due to TDP-43 overexpression. All comparisons were performed with similarly aged flies. (I–K) Overexpression of prosβ, Hsp70 or p35 alone does not lead to visible eye phenotypes.
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