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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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Yeast plasmid overexpression screen highlights the role of stress granules in TDP-43 toxicitya) Histogram showing the functional categories of genes (GO term process) with the relative frequency of genes from the TDP-43 overexpression screen compared to the yeast genome and to the FUS overexpression screen 33. Both the TDP-43 and FUS screens were enriched for RNA metabolic process genes, while the TDP-43 screen was enriched for cell cycle, transport, and protein modification process genes.b) While the TDP-43 and FUS screens were both enriched for RNA metabolic process genes, most of the hits did not overlap with the exception of three genes.c) Stress granule genes identified in the screen enhance or suppress TDP-43 toxicity. Spotting assay showing that on the galactose plate (expression ON) the co-expression of HRP1, KEM1, or PBP1 with TDP-43 leads to enhanced toxicity (reduced growth) while the co-expression of TIS11 or VTS1 with TDP-43 leads to suppression of toxicity (increased growth).d) RNA-binding protein focused yeast interaction network for TDP-43 screen hits reveal connections to PAB1 and EIF2A homolog. Ten out of forty yeast genes that modified TDP-43 toxicity when overexpressed are annotated as RNA-binding proteins. These are displayed as black circles. GeneMANIA 56 was used to search for interacting genes based physical and genetic interactions. Interacting genes are displayed as grey circles and network edges are colored based on the type of interaction (red=physical interaction and green=genetic interaction). This analysis revealed strong connections to PAB1 and YGR054W, which are highlighted in blue. YGR054W is the yeast homolog of a human translation initiation factor EIF2A.e) GFP-tagged ALS-linked disease gene TDP-43 forms aggregates when expressed in yeast that colocalize with the stress granule marker PUB1-mCherry. Scale bar, 5 μm.
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Figure 1: Yeast plasmid overexpression screen highlights the role of stress granules in TDP-43 toxicitya) Histogram showing the functional categories of genes (GO term process) with the relative frequency of genes from the TDP-43 overexpression screen compared to the yeast genome and to the FUS overexpression screen 33. Both the TDP-43 and FUS screens were enriched for RNA metabolic process genes, while the TDP-43 screen was enriched for cell cycle, transport, and protein modification process genes.b) While the TDP-43 and FUS screens were both enriched for RNA metabolic process genes, most of the hits did not overlap with the exception of three genes.c) Stress granule genes identified in the screen enhance or suppress TDP-43 toxicity. Spotting assay showing that on the galactose plate (expression ON) the co-expression of HRP1, KEM1, or PBP1 with TDP-43 leads to enhanced toxicity (reduced growth) while the co-expression of TIS11 or VTS1 with TDP-43 leads to suppression of toxicity (increased growth).d) RNA-binding protein focused yeast interaction network for TDP-43 screen hits reveal connections to PAB1 and EIF2A homolog. Ten out of forty yeast genes that modified TDP-43 toxicity when overexpressed are annotated as RNA-binding proteins. These are displayed as black circles. GeneMANIA 56 was used to search for interacting genes based physical and genetic interactions. Interacting genes are displayed as grey circles and network edges are colored based on the type of interaction (red=physical interaction and green=genetic interaction). This analysis revealed strong connections to PAB1 and YGR054W, which are highlighted in blue. YGR054W is the yeast homolog of a human translation initiation factor EIF2A.e) GFP-tagged ALS-linked disease gene TDP-43 forms aggregates when expressed in yeast that colocalize with the stress granule marker PUB1-mCherry. Scale bar, 5 μm.

Mentions: We previously developed a TDP-43 yeast model that recapitulates key features of TDP-43 associated with disease including toxicity and aggregation 29,30. Using this model, we performed a screen for genes that when upregulated would modify TDP-43 toxicity 25,29–31. A yeast query strain carrying an integrated copy of TDP-43 under the control of a galactose inducible promoter was transformed with the yeast FLEX gene library, which contains an arrayed set of 5500 genes that are overexpressed upon galactose induction 24,32. Transformed yeast cells were subsequently plated on glucose (TDP-43 expression OFF) and galactose plates (TDP-43 expression ON), and screened for genes that suppressed or enhanced TDP-43 toxicity. We identified 40 hits, which included 13 suppressors and 27 enhancers of TDP-43 toxicity (Table 1). GO term analysis on the entire set of screen hits showed a striking enrichment in RNA metabolic process, cell cycle, transport and protein modification process genes (Fig. 1a). While there was little overlap between the hits found in this TDP-43 screen and in a previous FUS overexpression screen 33, both screens were enriched for RNA metabolic processes, and to a lesser extent cell cycle genes (Fig. 1a,b). We performed the screen in triplicate, and hits confirmed all three times were independently verified with more sensitive spotting assays (Fig. 1c).


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)

Yeast plasmid overexpression screen highlights the role of stress granules in TDP-43 toxicitya) Histogram showing the functional categories of genes (GO term process) with the relative frequency of genes from the TDP-43 overexpression screen compared to the yeast genome and to the FUS overexpression screen 33. Both the TDP-43 and FUS screens were enriched for RNA metabolic process genes, while the TDP-43 screen was enriched for cell cycle, transport, and protein modification process genes.b) While the TDP-43 and FUS screens were both enriched for RNA metabolic process genes, most of the hits did not overlap with the exception of three genes.c) Stress granule genes identified in the screen enhance or suppress TDP-43 toxicity. Spotting assay showing that on the galactose plate (expression ON) the co-expression of HRP1, KEM1, or PBP1 with TDP-43 leads to enhanced toxicity (reduced growth) while the co-expression of TIS11 or VTS1 with TDP-43 leads to suppression of toxicity (increased growth).d) RNA-binding protein focused yeast interaction network for TDP-43 screen hits reveal connections to PAB1 and EIF2A homolog. Ten out of forty yeast genes that modified TDP-43 toxicity when overexpressed are annotated as RNA-binding proteins. These are displayed as black circles. GeneMANIA 56 was used to search for interacting genes based physical and genetic interactions. Interacting genes are displayed as grey circles and network edges are colored based on the type of interaction (red=physical interaction and green=genetic interaction). This analysis revealed strong connections to PAB1 and YGR054W, which are highlighted in blue. YGR054W is the yeast homolog of a human translation initiation factor EIF2A.e) GFP-tagged ALS-linked disease gene TDP-43 forms aggregates when expressed in yeast that colocalize with the stress granule marker PUB1-mCherry. Scale bar, 5 μm.
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Related In: Results  -  Collection

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Figure 1: Yeast plasmid overexpression screen highlights the role of stress granules in TDP-43 toxicitya) Histogram showing the functional categories of genes (GO term process) with the relative frequency of genes from the TDP-43 overexpression screen compared to the yeast genome and to the FUS overexpression screen 33. Both the TDP-43 and FUS screens were enriched for RNA metabolic process genes, while the TDP-43 screen was enriched for cell cycle, transport, and protein modification process genes.b) While the TDP-43 and FUS screens were both enriched for RNA metabolic process genes, most of the hits did not overlap with the exception of three genes.c) Stress granule genes identified in the screen enhance or suppress TDP-43 toxicity. Spotting assay showing that on the galactose plate (expression ON) the co-expression of HRP1, KEM1, or PBP1 with TDP-43 leads to enhanced toxicity (reduced growth) while the co-expression of TIS11 or VTS1 with TDP-43 leads to suppression of toxicity (increased growth).d) RNA-binding protein focused yeast interaction network for TDP-43 screen hits reveal connections to PAB1 and EIF2A homolog. Ten out of forty yeast genes that modified TDP-43 toxicity when overexpressed are annotated as RNA-binding proteins. These are displayed as black circles. GeneMANIA 56 was used to search for interacting genes based physical and genetic interactions. Interacting genes are displayed as grey circles and network edges are colored based on the type of interaction (red=physical interaction and green=genetic interaction). This analysis revealed strong connections to PAB1 and YGR054W, which are highlighted in blue. YGR054W is the yeast homolog of a human translation initiation factor EIF2A.e) GFP-tagged ALS-linked disease gene TDP-43 forms aggregates when expressed in yeast that colocalize with the stress granule marker PUB1-mCherry. Scale bar, 5 μm.
Mentions: We previously developed a TDP-43 yeast model that recapitulates key features of TDP-43 associated with disease including toxicity and aggregation 29,30. Using this model, we performed a screen for genes that when upregulated would modify TDP-43 toxicity 25,29–31. A yeast query strain carrying an integrated copy of TDP-43 under the control of a galactose inducible promoter was transformed with the yeast FLEX gene library, which contains an arrayed set of 5500 genes that are overexpressed upon galactose induction 24,32. Transformed yeast cells were subsequently plated on glucose (TDP-43 expression OFF) and galactose plates (TDP-43 expression ON), and screened for genes that suppressed or enhanced TDP-43 toxicity. We identified 40 hits, which included 13 suppressors and 27 enhancers of TDP-43 toxicity (Table 1). GO term analysis on the entire set of screen hits showed a striking enrichment in RNA metabolic process, cell cycle, transport and protein modification process genes (Fig. 1a). While there was little overlap between the hits found in this TDP-43 screen and in a previous FUS overexpression screen 33, both screens were enriched for RNA metabolic processes, and to a lesser extent cell cycle genes (Fig. 1a,b). We performed the screen in triplicate, and hits confirmed all three times were independently verified with more sensitive spotting assays (Fig. 1c).

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