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Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.

Kim HJ, Raphael AR, LaDow ES, McGurk L, Weber RA, Trojanowski JQ, Lee VM, Finkbeiner S, Gitler AD, Bonini NM - Nat. Genet. (2013)

Bottom Line: A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules.In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis.These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. [2].

ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2α phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2α phosphorylation in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

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Genes that impact stress granule formation modulate TDP-43 toxicitya) TDP-43 expression increases eIF2α-phosphorylation levels. Genes were expressed in the nervous system in a drug-inducible manner with the elavGS driver. eIF2α- phosphorylation level of elavGS/UAS-YFP and elavGS, TDP-43/UAS-YFP flies fed as adults on RU486 (40μg/ml), and assessed at the indicated time-points. Genotypes: Control is elavGS/UAS-YFP. TDP-43 is elavGS, UAS-TDP-43(S)/UAS-YFP. Mean ± s.e.m., n=3 independent experiments. *p<0.05, *** p<0.001, n.s., not significant. (Student’s t-test).b) Altering the levels of genes that reduce stress granule formation mitigates TDP-43 toxicity, and that promote stress granule formation enhances TDP-43 toxicity. Mean ± 95% CI of four experiments. Genotypes: elavGS/UAS-YFP is elavGS/UAS-YFP. elavGS, TDP-43/UAS-YFP is elavGS, UAS-TDP-43(S)/UAS-YFP. elavGS, TDP-43/Gadd34 RNAi is elavGS, UAS-TDP-43(S)/UAS-Gadd34. RNAiHMS00811. elavGS, TDP-43/Rox8 RNAi is elavGS, UAS-TDP-43(S)/UAS-Rox8. RNAiHMS00472. elavGS, TDP-43/PEK RNAi is elavGS, UAS-TDP-43(S)/UAS-PEK. RNAiGL00030. All flies raised with RU486 (40μg/ml) (125 flies per genotype). ANOVA for significance, followed by Tukey’s multiple comparison test, **p<0.01, *** p<0.001, # p<0.0001, n.s., not significant.c) eIF2α-phosphorylation level of elavGS, TDP-43/UAS-YFP, elavGS, TDP-43/Rox8 RNAi, elavGS, TDP-43/Gadd34 RNAi and elavGS, TDP-43/PEK RNAi. Mean ± s.e.m., n=3 independent experiments. *p<0.05, n.s., not significant (Student’s t-test).d) Total, nuclear and cytosolic TDP-43 protein level in 10d fly heads. Genes predicted to increase stress granules formation increase cytoplasmic TDP-43 protein levels. Mean ± s.e.m., n=3 independent experiments. *** p<0.001, n.s., not significant (Student’s t-test).
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Figure 2: Genes that impact stress granule formation modulate TDP-43 toxicitya) TDP-43 expression increases eIF2α-phosphorylation levels. Genes were expressed in the nervous system in a drug-inducible manner with the elavGS driver. eIF2α- phosphorylation level of elavGS/UAS-YFP and elavGS, TDP-43/UAS-YFP flies fed as adults on RU486 (40μg/ml), and assessed at the indicated time-points. Genotypes: Control is elavGS/UAS-YFP. TDP-43 is elavGS, UAS-TDP-43(S)/UAS-YFP. Mean ± s.e.m., n=3 independent experiments. *p<0.05, *** p<0.001, n.s., not significant. (Student’s t-test).b) Altering the levels of genes that reduce stress granule formation mitigates TDP-43 toxicity, and that promote stress granule formation enhances TDP-43 toxicity. Mean ± 95% CI of four experiments. Genotypes: elavGS/UAS-YFP is elavGS/UAS-YFP. elavGS, TDP-43/UAS-YFP is elavGS, UAS-TDP-43(S)/UAS-YFP. elavGS, TDP-43/Gadd34 RNAi is elavGS, UAS-TDP-43(S)/UAS-Gadd34. RNAiHMS00811. elavGS, TDP-43/Rox8 RNAi is elavGS, UAS-TDP-43(S)/UAS-Rox8. RNAiHMS00472. elavGS, TDP-43/PEK RNAi is elavGS, UAS-TDP-43(S)/UAS-PEK. RNAiGL00030. All flies raised with RU486 (40μg/ml) (125 flies per genotype). ANOVA for significance, followed by Tukey’s multiple comparison test, **p<0.01, *** p<0.001, # p<0.0001, n.s., not significant.c) eIF2α-phosphorylation level of elavGS, TDP-43/UAS-YFP, elavGS, TDP-43/Rox8 RNAi, elavGS, TDP-43/Gadd34 RNAi and elavGS, TDP-43/PEK RNAi. Mean ± s.e.m., n=3 independent experiments. *p<0.05, n.s., not significant (Student’s t-test).d) Total, nuclear and cytosolic TDP-43 protein level in 10d fly heads. Genes predicted to increase stress granules formation increase cytoplasmic TDP-43 protein levels. Mean ± s.e.m., n=3 independent experiments. *** p<0.001, n.s., not significant (Student’s t-test).

Mentions: To test the significance of the interaction between stress granules and ALS-associated RNA binding protein toxicity in the nervous system, we used Drosophila. Although it is challenging to image stress granules in vivo in flies, eIF2α-phosphorylation induces the accumulation of non-functional translation initiation complexes that concentrate in stress granules, thus levels of eIF2α-phosphorylation are directly correlated with the levels of stress granules 13. We extracted protein from heads of control and TDP-43-expressing animals, and immunoblotted extracts with a phospho-specific eIF2α antibody (Ser51). This approach revealed a progressive increase in eIF2α-phosphorylation upon expression of TDP-43 in the brain: there was no difference between control and TDP-43-expressing flies in the levels of eIF2α-phosphorylation at 5d, but by 8d and 14d, the levels of eIF2α-phosphorylation were significantly increased to 1.4±0.1-fold (s.e.m.), and 1.6±0.1-fold (s.e.m.), respectively (Fig. 2a). There was no observed increase in levels of eIF2α protein, showing that the change in phospho-eIF2α levels represented a change in the stress granule specific form (Supplementary Fig. 1). These data suggest that TDP-43 expression in the fly brain induces chronic eIF2α-phosphorylation. Moreover, the increase in eIF2α-phosphorylation indicates a state of prolonged translational repression 22.


Therapeutic modulation of eIF2α phosphorylation rescues TDP-43 toxicity in amyotrophic lateral sclerosis disease models.

Kim HJ, Raphael AR, LaDow ES, McGurk L, Weber RA, Trojanowski JQ, Lee VM, Finkbeiner S, Gitler AD, Bonini NM - Nat. Genet. (2013)

Genes that impact stress granule formation modulate TDP-43 toxicitya) TDP-43 expression increases eIF2α-phosphorylation levels. Genes were expressed in the nervous system in a drug-inducible manner with the elavGS driver. eIF2α- phosphorylation level of elavGS/UAS-YFP and elavGS, TDP-43/UAS-YFP flies fed as adults on RU486 (40μg/ml), and assessed at the indicated time-points. Genotypes: Control is elavGS/UAS-YFP. TDP-43 is elavGS, UAS-TDP-43(S)/UAS-YFP. Mean ± s.e.m., n=3 independent experiments. *p<0.05, *** p<0.001, n.s., not significant. (Student’s t-test).b) Altering the levels of genes that reduce stress granule formation mitigates TDP-43 toxicity, and that promote stress granule formation enhances TDP-43 toxicity. Mean ± 95% CI of four experiments. Genotypes: elavGS/UAS-YFP is elavGS/UAS-YFP. elavGS, TDP-43/UAS-YFP is elavGS, UAS-TDP-43(S)/UAS-YFP. elavGS, TDP-43/Gadd34 RNAi is elavGS, UAS-TDP-43(S)/UAS-Gadd34. RNAiHMS00811. elavGS, TDP-43/Rox8 RNAi is elavGS, UAS-TDP-43(S)/UAS-Rox8. RNAiHMS00472. elavGS, TDP-43/PEK RNAi is elavGS, UAS-TDP-43(S)/UAS-PEK. RNAiGL00030. All flies raised with RU486 (40μg/ml) (125 flies per genotype). ANOVA for significance, followed by Tukey’s multiple comparison test, **p<0.01, *** p<0.001, # p<0.0001, n.s., not significant.c) eIF2α-phosphorylation level of elavGS, TDP-43/UAS-YFP, elavGS, TDP-43/Rox8 RNAi, elavGS, TDP-43/Gadd34 RNAi and elavGS, TDP-43/PEK RNAi. Mean ± s.e.m., n=3 independent experiments. *p<0.05, n.s., not significant (Student’s t-test).d) Total, nuclear and cytosolic TDP-43 protein level in 10d fly heads. Genes predicted to increase stress granules formation increase cytoplasmic TDP-43 protein levels. Mean ± s.e.m., n=3 independent experiments. *** p<0.001, n.s., not significant (Student’s t-test).
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Figure 2: Genes that impact stress granule formation modulate TDP-43 toxicitya) TDP-43 expression increases eIF2α-phosphorylation levels. Genes were expressed in the nervous system in a drug-inducible manner with the elavGS driver. eIF2α- phosphorylation level of elavGS/UAS-YFP and elavGS, TDP-43/UAS-YFP flies fed as adults on RU486 (40μg/ml), and assessed at the indicated time-points. Genotypes: Control is elavGS/UAS-YFP. TDP-43 is elavGS, UAS-TDP-43(S)/UAS-YFP. Mean ± s.e.m., n=3 independent experiments. *p<0.05, *** p<0.001, n.s., not significant. (Student’s t-test).b) Altering the levels of genes that reduce stress granule formation mitigates TDP-43 toxicity, and that promote stress granule formation enhances TDP-43 toxicity. Mean ± 95% CI of four experiments. Genotypes: elavGS/UAS-YFP is elavGS/UAS-YFP. elavGS, TDP-43/UAS-YFP is elavGS, UAS-TDP-43(S)/UAS-YFP. elavGS, TDP-43/Gadd34 RNAi is elavGS, UAS-TDP-43(S)/UAS-Gadd34. RNAiHMS00811. elavGS, TDP-43/Rox8 RNAi is elavGS, UAS-TDP-43(S)/UAS-Rox8. RNAiHMS00472. elavGS, TDP-43/PEK RNAi is elavGS, UAS-TDP-43(S)/UAS-PEK. RNAiGL00030. All flies raised with RU486 (40μg/ml) (125 flies per genotype). ANOVA for significance, followed by Tukey’s multiple comparison test, **p<0.01, *** p<0.001, # p<0.0001, n.s., not significant.c) eIF2α-phosphorylation level of elavGS, TDP-43/UAS-YFP, elavGS, TDP-43/Rox8 RNAi, elavGS, TDP-43/Gadd34 RNAi and elavGS, TDP-43/PEK RNAi. Mean ± s.e.m., n=3 independent experiments. *p<0.05, n.s., not significant (Student’s t-test).d) Total, nuclear and cytosolic TDP-43 protein level in 10d fly heads. Genes predicted to increase stress granules formation increase cytoplasmic TDP-43 protein levels. Mean ± s.e.m., n=3 independent experiments. *** p<0.001, n.s., not significant (Student’s t-test).
Mentions: To test the significance of the interaction between stress granules and ALS-associated RNA binding protein toxicity in the nervous system, we used Drosophila. Although it is challenging to image stress granules in vivo in flies, eIF2α-phosphorylation induces the accumulation of non-functional translation initiation complexes that concentrate in stress granules, thus levels of eIF2α-phosphorylation are directly correlated with the levels of stress granules 13. We extracted protein from heads of control and TDP-43-expressing animals, and immunoblotted extracts with a phospho-specific eIF2α antibody (Ser51). This approach revealed a progressive increase in eIF2α-phosphorylation upon expression of TDP-43 in the brain: there was no difference between control and TDP-43-expressing flies in the levels of eIF2α-phosphorylation at 5d, but by 8d and 14d, the levels of eIF2α-phosphorylation were significantly increased to 1.4±0.1-fold (s.e.m.), and 1.6±0.1-fold (s.e.m.), respectively (Fig. 2a). There was no observed increase in levels of eIF2α protein, showing that the change in phospho-eIF2α levels represented a change in the stress granule specific form (Supplementary Fig. 1). These data suggest that TDP-43 expression in the fly brain induces chronic eIF2α-phosphorylation. Moreover, the increase in eIF2α-phosphorylation indicates a state of prolonged translational repression 22.

Bottom Line: A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules.In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis.These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Biology, University of Pennsylvania, Philadelphia, Pennsylvania, USA. [2].

ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal, late-onset neurodegenerative disease primarily affecting motor neurons. A unifying feature of many proteins associated with ALS, including TDP-43 and ataxin-2, is that they localize to stress granules. Unexpectedly, we found that genes that modulate stress granules are strong modifiers of TDP-43 toxicity in Saccharomyces cerevisiae and Drosophila melanogaster. eIF2α phosphorylation is upregulated by TDP-43 toxicity in flies, and TDP-43 interacts with a central stress granule component, polyA-binding protein (PABP). In human ALS spinal cord neurons, PABP accumulates abnormally, suggesting that prolonged stress granule dysfunction may contribute to pathogenesis. We investigated the efficacy of a small molecule inhibitor of eIF2α phosphorylation in ALS models. Treatment with this inhibitor mitigated TDP-43 toxicity in flies and mammalian neurons. These findings indicate that the dysfunction induced by prolonged stress granule formation might contribute directly to ALS and that compounds that mitigate this process may represent a novel therapeutic approach.

Show MeSH
Related in: MedlinePlus