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. 2012 Jul;8(7):e1002806.
doi: 10.1371/journal.pgen.1002806. Epub 2012 Jul 5.

TDP-1/TDP-43 regulates stress signaling and age-dependent proteotoxicity in Caenorhabditis elegans

Alexandra Vaccaro  1 , Arnaud Tauffenberger, Peter E A Ash, Yari Carlomagno, Leonard Petrucelli, J Alex Parker
Affiliations

TDP-1/TDP-43 regulates stress signaling and age-dependent proteotoxicity in Caenorhabditis elegans

Alexandra Vaccaro et al. PLoS Genet. 2012 Jul.

Abstract

TDP-43 is a multifunctional nucleic acid binding protein linked to several neurodegenerative diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia. To learn more about the normal biological and abnormal pathological role of this protein, we turned to Caenorhabditis elegans and its orthologue TDP-1. We report that TDP-1 functions in the Insulin/IGF pathway to regulate longevity and the oxidative stress response downstream from the forkhead transcription factor DAF-16/FOXO3a. However, although tdp-1 mutants are stress-sensitive, chronic upregulation of tdp-1 expression is toxic and decreases lifespan. ALS-associated mutations in TDP-43 or the related RNA binding protein FUS activate the unfolded protein response and generate oxidative stress leading to the daf-16-dependent upregulation of tdp-1 expression with negative effects on neuronal function and lifespan. Consistently, deletion of endogenous tdp-1 rescues mutant TDP-43 and FUS proteotoxicity in C. elegans. These results suggest that chronic induction of wild-type TDP-1/TDP-43 by cellular stress may propagate neurodegeneration and decrease lifespan.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. tdp-1 regulates lifespan.
(A) Mutation in tdp-1 reduced the lifespan of daf-2(e1370) mutants but did not further reduce the lifespan of daf-16(mu86) mutants. (B) RNAi against tdp-1 reduced the lifespan of daf-2(e1370) mutants. EV is the empty vector control. (C) tdp-1(ok803) mutants had increased lifespan compared to N2 worms at 20°C. (D) tdp-1(ok803) mutants and N2 worms had comparable lifespan at 25°C. (E) TDP-1::GFP overexpression strains had reduced lifespans compared to N2 worms or tdp-1(ok803) mutants at 20°C. (F) TDP-1::GFP transgenic had reduced lifespans compared to N2 worms when grown at 25°C. Please also see Table S1.
Figure 2
Figure 2. tdp-1 specifies stress response signaling.
(A) tdp-1(ok803) mutants were more sensitive to juglone than N2 worms (P<0.001). daf-2(e1370) mutants were completely resistant to juglone-induced mortality after 14 hours of exposure. daf-2(e1370);tdp-1(ok803) mutants were highly sensitive to juglone toxicity compared to N2 or daf-2(e1370) controls (P<0.001). (B) tdp-1(ok803) mutants were more sensitive to hydrogen peroxide than N2 worms (P<0.001). daf-2(e1370) mutants were more resistant to hydrogen peroxide-induced mortality after 14 hours of exposure than N2 worms (P<0.001). daf-2(e1370);tdp-1(ok803) mutants were highly sensitive to hydrogen peroxide toxicity compared to N2 or daf-2(e1370) controls (P<0.001). (C) tdp-1(ok803) mutants were more sensitive to high NaCl levels than N2 worms (P<0.001), while daf-2(e1370) and daf-2(e1370);tdp-1(ok803) mutants were more resistant to NaCl-induced mortality than N2 worms after 14 hours of exposure (P<0.001). (D) tdp-1(ok803) mutants were more sensitive to high sorbitol levels than N2 worms (P<0.001), while daf-2(e1370) and daf-2(e1370);tdp-1(ok803) mutants were completely resistant to Sorbitol-induced mortality after 14 hours of exposure (P<0.001). (E) RT-PCR revealed no change in the expression of the catalases ctl-1 and ctl-2 by deletion of tdp-1. Expression of the superoxide dismutase sod-3 was diminished in daf-2(e1370);tdp-1(ok803) mutants. No expression of tdp-1 was observed in tdp-1(ok803) or daf-2(e1370);tdp-1(ok803) mutants. act-3 was used as an endogenous control. Please see Figure S2 and Table S2.
Figure 3
Figure 3. Insulin/IGF signaling and cellular stress regulate TDP-1 expression.
(A) Fluorescent signals observed under low magnification in an adult transgenic worm expressing a full-length TDP-1::GFP fusion under control of the endogenous tdp-1 promoter. The head (h) and tail (t) of the animal are indicated. GFP is observed in many tissues and appears to be primarily nuclear. (B) High magnification image of the head of a TDP-1::GFP transgenic revealing expression in head neurons (n) as well as the intestine (i). (C) High magnification image of the tail region of a TDP-1::GFP transgenic revealing expression in tail neurons (n) as well as muscle cells (m). (D) High magnification image revealing the expression of TDP-1::GFP in intestinal (i) cells. (E–I) are low-resolution photographs of young adult TDP-1::GFP transgenic worms. The images have been taken at the same exposure, converted to black and white and photo-reversed to aid visualization. Difficult to see worms are outlined. (E) TDP-1::GFP is widely expressed and primarily nuclear under normal conditions. TDP-1::GFP expression was highly induced by osmotic stress with NaCl or oxidative stress with juglone compared to untreated worms as seen in the left panel. (F) TDP-1::GFP expression was observed in tdp-1(ok803) mutants and was induced by osmotic and oxidative stress. (G) Compared to untreated controls in (A) TDP-1::GFP expression was elevated in daf-2(e1370) mutants and further upregulated by stress conditions. (H) TDP-1::GFP remained lowly expressed in daf-2(e1368) mutants under normal and stress conditions. (I) TDP-1::GFP showed faint expression and nuclear localization in daf-16(mu86) mutants. Osmotic stress induced TDP-1::GFP expression in daf-16(mu86) mutants. TDP-1::GFP was not induced by oxidative stress in daf-16(mu86) mutants. (J) TDP-1::GFP is primarily expressed in the nuclei (n, marked with DAPI) of non-stressed transgenics. (K) TDP-1::GFP expression is increased and is now also found in the cytoplasm (c) of daf-2 animals. Please also see Figure S4.
Figure 4
Figure 4. Endogenous TDP-1 protein levels influenced by stress and Insulin/IGF signaling.
Western blotting of worm extracts from different strains under normal and stress conditions with an anti-TDP-1 antibody. (A) Osmotic and oxidative stress significantly increased the expression of TDP-1 in N2 worms. (B) The level of TDP-1 protein was comparable in untreated and osmotically stressed daf-2(e1370) worms. TDP-1 protein expression was significantly increased by exposure to oxidative stress in daf-2(e1370) worms. (C) TDP-1 protein levels remained low in daf-2(e1368) animals with or without stress. (D) TDP-1 protein expression was significantly increased by osmotic stress in daf-16(mu86) animals. Oxidative stress did not induce TDP-1 expression in daf-16 mutants.
Figure 5
Figure 5. Mutant TDP-43 induces HSP-4/BiP expression.
(A to D) are low-resolution photographs of young adult transgenic worms. The images have been converted to black and white and photo-reversed to aid visualization. Difficult to see worms are outlined. (A) Representative image of a young adult worm containing an integrated hsp-4p::GFP transgene under non-stressed conditions. (B) hsp-4p::GFP expression was induced by the chemical ER stressor tunicamycin. (C) hsp-4p::GFP expression was not induced by wild type TDP-43 in a double transgenic TDP-43 WT;hsp-4p::GFP strain. (D) hsp-4p::GFP expression was induced by mutant TDP-43 in a double transgenic TDP-43[A315T];hsp-4p::GFP strain.
Figure 6
Figure 6. TDP-1 expression is induced by proteotoxicity.
(A–G) are low-resolution photographs of young adult transgenic worms. The images have been converted to black and white and photo-reversed to aid visualization. Difficult to see worms are outlined. (A) Low expression and nuclear localization of TDP-1::GFP under normal growth conditions. (B) The ER stressor tunicamycin highly induced TDP-1::GFP expression. (C) Wild type TDP-43 did not induce TDP-1::GFP expression in a TDP-1::GFP;TDP-43 WT transgenic strain. (D) Mutant TDP-43 induced TDP-1::GFP expression in a TDP-1::GFP;TDP-43[A315T] transgenic strain. (E) Wild type FUS did not induce TDP-1::GFP expression in a TDP-1::GFP;FUS WT transgenic strain. (F) Mutant FUS induced TDP-1::GFP expression in a TDP-1::GFP;FUS[S57Δ] transgenic strain. (G) Mutation in daf-16 blocked the induction of TDP-1::GFP expression by tunicamycin treatment. Please also see Figure S4.
Figure 7
Figure 7. Mutant TDP-43 and FUS increase oxidative stress.
(A–D) are low-resolution images of young adult transgenic worms stained with dihydrofluorescein diacetate. Transgenics expressing wild type (A) TDP-43 or (B) wild type FUS showed no fluorescence compared to the bright fluorescent signals observed in animals expressing (C) mutant TDP-43 or (D) mutant FUS. (E to F) are higher magnification images of DAF-16::GFP transgenics. (E) Diffuse appearance of predominantly cytoplasmic DAF-16::GFP. (F) Introduction of mutant TDP-43 causes nuclear localization (arrows) of DAF-16::GFP.
Figure 8
Figure 8. Opposing effects of daf-2 on proteotoxicity.
(A) daf-2(e1368);TDP-43[A315T] animals had significantly deceased rates of paralysis, while daf-2(e1370);TDP-43[A315T] animals had enhanced paralysis compared to TDP-43[A315T] transgenics alone (P<0.001). (B) daf-2(e1368);TDP-43[A315T] animals had significantly deceased motor neuron degeneration, while daf-2(e1370);TDP-43[A315T] animals had enhanced neurodegeneration compared to TDP-43[A315T] transgenics alone (*P<0.001, **P<0.05). (C) Western blotting revealed a significant reduction in the amount of insoluble TDP-43 protein in extracts from daf-2(e1368);TDP-43[A315T] animals compared to TDP-43 alone. daf-2(e1370) had no effect on mutant TDP-43 solubility. Please also see Table S3.
Figure 9
Figure 9. TDP-1 regulates mutant TDP-43 and FUS proteotoxicity in C. elegans.
(A) TDP-43[A315T];TDP-1::GFP transgenics had shorter lifespans than transgenics expressing mutant TDP-43 alone (P<0.01). (B) FUS[S57Δ];TDP-1::GFP transgenics had shorter lifespans than transgenics expressing mutant FUS alone (P<0.001). Transgenics expressing (C) mutant TDP-43 or (D) mutant FUS along with TDP-1::GFP had accelerated rates of paralysis compared to transgenics expressing either mutant TDP-43 or mutant FUS alone (P<0.001 for mutant TDP-43 or FUS compared to mutant TDP-43;TDP-1::GFP or mutant FUS;TDP-1::GFP respectively). (E) Transgenics expressing mutant TDP-43 or FUS show age-dependent paralysis that is greatly reduced in worms harboring a deletion mutation of endogenous tdp-1 (P<0.001). (F) Age-dependent motor neuron degeneration was reduced in tdp-1(ok803);TDP-43[A315T] and tdp-1(ok803);FUS[S57Δ] strains compared to TDP-43[A315T] or FUS[S57Δ] transgenics respectively (*, **P<0.001). Deletion of tdp-1 did not affect the proportion of insoluble (G) mutant TDP-43 or (H) FUS proteins in extracts from whole worms. Please also see Tables S1 and S3.
Figure 10
Figure 10. Integrated model for stress-induced TDP-1 expression.
TDP-1 has multiple contributions to lifespan and the cellular stress response. (A) Under normal conditions TDP-1 may function in the Insulin/IGFP pathway upstream from DAF-16 to regulate lifespan. (B) A variety of cellular stresses induce TDP-1 expression. TDP-1 induction by oxidative stress and/or the Insulin/IGF pathway is dependent on DAF-16, while induction by osmotic stress is independent of DAF-16. Misfolded proteins activate the unfolded protein response and a secondary consequence of this is the generation of oxidative stress that in turn can induce TDP-1 expression via DAF-16. The downstream consequences of tdp-1 expression are dependent on the length and strength of induction. tdp-1 mutants are sensitive to stress suggesting that TDP-1 is essential for protection against acute stress. Genetically encoded proteotoxicity from proteins like mutant TDP-43 leads to chronic induction of TDP-1 expression with negative consequences including enhanced neurodegeneration. This model also suggests that mutant proteins may act as a seed for the induction of pathological TDP-1 expression.
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References

    1. Lagier-Tourenne C, Polymenidou M, Cleveland DW. TDP-43 and FUS/TLS: emerging roles in RNA processing and neurodegeneration. Hum Mol Genet. 2010;19:R46–64. - PMC - PubMed
    1. Anderson P, Kedersha N. Stress granules. Curr Biol. 2009;19:R397–398. - PubMed
    1. Anderson P, Kedersha N. RNA granules: post-transcriptional and epigenetic modulators of gene expression. Nat Rev Mol Cell Biol. 2009;10:430–436. - PubMed
    1. Kenyon C. The plasticity of aging: insights from long-lived mutants. Cell. 2005;120:449–460. - PubMed
    1. Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. A C. elegans mutant that lives twice as long as wild type. Nature. 1993;366:461–464. - PubMed

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