TDP-43-mediated neuron loss in vivo requires RNA-binding activity - PubMed (original) (raw)

TDP-43-mediated neuron loss in vivo requires RNA-binding activity

Aaron Voigt et al. PLoS One. 2010.

Abstract

Alteration and/or mutations of the ribonucleoprotein TDP-43 have been firmly linked to human neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). The relative impacts of TDP-43 alteration, mutation, or inherent protein function on neural integrity, however, remain less clear--a situation confounded by conflicting reports based on transient and/or random-insertion transgenic expression. We therefore performed a stringent comparative investigation of impacts of these TDP-43 modifications on neural integrity in vivo. To achieve this, we systematically screened ALS/FTLD-associated and synthetic TDP-43 isoforms via same-site gene insertion and neural expression in Drosophila; followed by transposon-based motor neuron-specific transgenesis in a chick vertebrate system. Using this bi-systemic approach we uncovered a requirement of inherent TDP-43 RNA-binding function--but not ALS/FTLD-linked mutation, mislocalization, or truncation--for TDP-43-mediated neurotoxicity in vivo.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. Analysis of different TDP-43 variants used for ectopic expression.

(A) Schematic overview of TDP-43 variants tested in vivo. Positions and nature of amino acid substitutions and size of two C-terminal fragments (CTFs) analyzed are indicated. Synthetic mutations (SM) were introduced to interfere with TDP-43 localization or function. Mutations in the nuclear localization signal (ΔNLS) were introduced to target TDP-43 to the cytoplasm and mutations F147L/F149L (FFLL) in the first RNA recognition motive (RRM1) were introduced to impair TDP-43 inherent RNA-binding capacity. (B) Loss of RNA-binding by TDP-43FFLL. Biotinylated oligonucleotides were incubated with lysates from either untransfected HEK293 cells or HEK293 cells transfected with FLAG-tagged TDP-43WT or TDP-43FFLL (left, input) followed by UV crosslinking and streptavidin-pulldown of Biotin-RNA (right, pulldown). Precipitates were separated, blotted and membranes probed with FLAG- (upper panel) and TDP-43-specific (lower panel) antibodies to assay co-precipitation of TDP-43. Note: Co-precipitation of endogenous TDP-43 with (UG)12-repeats in all lysates. Ectopic FLAG-TDP-43WT, but not FLAG-TDP-43FFLL co-precipitated with cognate UG repeats. GAPDH served as loading control for protein input. (C) Equalized pan-neural expression of _φ-C31_-inserted TDP-43 variants in adult Drosophilae. TDP-43 expression was visualized using an anti-human TDP-43 antibody. Syntaxin served as loading control. (D) Assessment of relative TBPH and TDP-43 expression levels. Left graph: Relative abundance of endogenous TBPH transcripts in relation to actin5C of wild type strain OregonR (OreR), the Gal4-driver line (elavC155::Gal4) and flies with pan-neural TDP-43 expression (elavC155::Gal4/Y;UAS::TDP-43WT/+). TBPH levels were not significantly different in analyzed genotypes. Right graph: mRNA abundance of TDP-43 in flies with pan-neural expression of the different TDP-43 variants normalized to actin5C and TBPH mRNA levels. Significant differences are indicated. *p<0.05; ***p<0.001. Head lysates of flies with pan-neural expression of TDP-43 were used for Western blot and qPCR analysis. elav::Gal4 flies without a TDP-43 transgene (elav) were used as negative control.

Figure 2

Figure 2. Localization of TDP-43 variants in human cells.

HEK293E cells transfected with N-terminal Flag-tagged TDP-43 variants detected with Flag- (left, green) and TDP-43 (middle, red) specific antibodies. Overlay (right) with Hoechst stained DNA (blue). (A) Exogenous TDP-43 had to be visualized via fused Flag-tag, as HEK cells show robust endogenous expression of TDP-43. (B) Ectopic TDP-43WT mainly localized to the nucleus. (C) In contrast, TDP-43ΔNLS exclusively localized to cytosol without showing nuclear Flag-signal. Similar to TDP-43FFLL (D), also TDP-43MS (E–I) displayed a nuclear localization. However, TDP-43FFLL localized in a characteristic punctuate pattern throughout the nucleus (D). (J) TDP-43CTF displayed a predominantly cytoplasmic localization similar to TDP-43ΔNLS (compare C and J). Scale bar indicates 10 µm.

Figure 3

Figure 3. Localization of TDP-43 in Drosophila melanogaster and Gallus gallus.

Confocal sections of eye imaginal discs from Drosophila larvae (left panel) and motor neurons from Gallus (right panel) expressing indicated TDP-43 variants. To be able to discriminate between the two in vivo systems, ectopic TDP-43 in Drosophila is shown in green, whereas TDP-43 in Gallus is shown in red. Subcellular localization of the different TDP-43 variants was found to be identical between fly and chick. TDP-43WT (A) localized mainly to the nucleus, while TDP-43ΔNLS (B) was found predominantly in the cytoplasm. TDP-43FFLL (C) and TDP-43A315T (D) displayed a nuclear distribution. Only cells with very high expression levels of usually nuclear TDP-43 displayed a detectable cytoplasmic staining (example in case of TDP-43FFLL marked by asterisk). DNA was stained with Sytox® Orange (fly, red) or DAPI (chick, white). Scale bar indicates 50 µm. Neuronal expression was mediated by elav::Gal4 (flies) or Hb9::Cre (chick).

Figure 4

Figure 4. Requirement for RNA-binding activity for TDP-43-mediated neural defects in Drosophila.

(A) TDP-43 expression reduces longevity in Drosophila. Flies with pan-neural (elav::Gal4) expression of indicated TDP-43 variants were assayed for longevity. (B) Median survival of respective survival curves. (C) Cross-comparison of survival curves with regard to statistical significance after Bonferroni correction. (D) Age-dependent locomotion defects after TDP-43 expression in motor neurons. Flies expressing indicated TDP-43 variants specifically in motor neurons (D42::Gal4) were assayed for negative geotaxis at 1, 10 and 20 days post eclosion. (E) Detailed summary of statistical analysis (2-Way ANOVA followed by Bonferroni post-hoc tests) of age-dependent locomotion effects. Genotypes of flies analyzed: elavC155::Gal4/Y;UAS::TDP-43/+ in longevity analysis and w/Y;UAS::TDP-43/+;D42::Gal4/+ in case of locomotion assay. elavC155::Gal4/Y (longevity) and w/Y;;D42::Gal4 (climbing) served as controls. *p<0.05; **p<0.01; ***p<0.001; ns not significant.

Figure 5

Figure 5. RNA-binding activity is required for TDP-43-mediated motor neuron loss in chick.

(A) Stable unilateral expression of human TDP-43 variants in chick (Gallus gallus) spinal cord. [i] Schematic of expression system mediating motor neuron-restricted expression upon unilateral transfection. [ii] Unilateral expression of human TDP-43FFLL (red) in embryonic day 9 (E9) chick spinal cord (nuclei labeled with DAPI: blue): transversal section at lumbar levels. “−” and “+” respectively indicate control and transfected hemicords. Isl1/2 labels motor neuron nuclei (green). (B) [i] Examples of thoracic motor columns (Isl1/2+ motor neurons: green) upon TDP-43 variant expression (red). [ii] Quantification of motor neuron loss upon TDP-43 variant expression over all obtained sections (in “-” versus “+” hemicord). Differences relative to control (t-student's test) are indicated. (C) [i] Activated Caspase-3 (green) detected in E5 motor neurons upon TDP-43WT expression (indicated by IRES-cherry bi-cistronic reporter: red). Compared to E5 motor neurons expressing TDP-43WT, little activation of Caspase-3 was detected upon TDP-43CTF expression. [ii] Quantification of Caspase-3 activation in motor neurons of transfected hemicords versus vector control. Significant differences are indicated (t-student's test - relative to control). *p<0.05; **p<0.01; ***p<0.001; ns not significant.

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