Identification of Genetic Modifiers of TDP-43 Neurotoxicity in Drosophila

<div><p>Cytosolic aggregation of the nuclear RNA-binding protein TDP-43 is a histopathologic signature of degenerating neurons in amyotrophic lateral sclerosis (ALS), and mutations in the <i>TARDBP</i> gene encoding TDP-43 cause dominantly inherited forms of this condition. To understand the relationship between TDP-43 misregulation and neurotoxicity, we and others have used Drosophila as a model system, in which overexpression of either wild-type TDP-43 or its ALS-associated mutants in neurons is sufficient to induce neurotoxicity, paralysis, and early death. Using microarrays, we have examined gene expression patterns that accompany TDP-43-induced neurotoxicity in the fly system. Constitutive expression of TDP-43 in the Drosophila compound eye elicited widespread gene expression changes, with strong upregulation of cell cycle regulatory genes and genes functioning in the Notch intercellular communication pathway. Inducible expression of TDP-43 specifically in neurons elicited significant expression differences in a more restricted set of genes. Genes that were upregulated in both paradigms included <i>SpindleB</i> and the Notch target <i>Hey</i>, which appeared to be a direct TDP-43 target. Mutations that diminished activity of Notch or disrupted the function of downstream Notch target genes extended the lifespan of TDP-43 transgenic flies, suggesting that Notch activation was deleterious in this model. Finally, we showed that mutation of the nucleoporin <i>Nup50</i> increased the lifespan of TDP-43 transgenic flies, suggesting that nuclear events contribute to TDP-43-dependent neurotoxicity. The combined findings identified pathways whose deregulation might contribute to TDP-43-induced neurotoxicity in Drosophila.</p> </div

Effects of Notch pathway knockdown on D42-Gal4/UAS-TDP-43 and control fly lifespan.

<p>(<b>A</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies crossed to mutants of <i>Delta</i> (<i>Dl</i>^RevF10) and <i>Serrate</i> (<i>Ser</i>^Rx82) or <i>Enhancer of split</i> (<i>E(spl)</i>^rv1). Median survival (days) of: D42-Gal4/UAS-TDP-43 = 24, N = 213; D42-Gal4/UAS-TDP-43, <i>Dl</i>^RevF10<i>Ser</i>^RX82 = 28, N = 215, p<0.0001; D42-Gal4/UAS-TDP-43, <i>E(spl)</i>^rv1 = 28, N = 220, p<0.0001. (<b>B</b>) Survival curves of D42-Gal4 control flies containing <i>Dl</i>^RevF10<i>Ser</i>^Rx82 and <i>E(spl)</i>^rv1 LOF alleles. Median survival (days) of: D42-Gal4/+ = 61, N = 221; D42-Gal4/<i>Dl</i>^RevF10, <i>Ser</i>^RX82 = 56, N = 219, p<0.0001; D42-Gal4/<i>E(spl)</i>^rv1 = 51, N = 216, p<0.0001; (<b>C</b> and <b>D</b>) TDP-43 expression level was not affected by the indicated mutations. D42-Gal4/UAS-TDP-43 flies were crossed to <i>w</i>1118 control flies or to the <i>Dl</i>^RevF10<i>Ser</i>^Rx82, <i>E(spl)</i>^rv1, <i>Nup50</i>^kg09557, ED1735 deletion line. Flies containing D42-Gal4/UAS-TDP-43 were heterozygous with each testing allele. Tubulin or Gal4 was used as a loading control; blots were representative of multiple experiments.</p

Gene expression changes in TDP-43 flies, arranged by category.

<p>All significantly changed Notch genes are shown, but only the most highly upregulated mitochondrial/redox and cell cycle genes are included.</p

Analysis of <i>Nup50</i> and other candidate genes.

<p>(<b>A</b>) Survival curves of D42-Gal4/UAS-TDP-43 crossed to <i>Pabp2</i> (<i>Pabp2</i>⁰¹or <i>Pabp2</i>^kg02359) and the <i>Rs1</i>^k09514 mutant lines. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 21, N = 110; D42-Gal4/UAS-TDP-43, <i>PABP</i>⁰¹ = 24, N = 63, p<0.0001; D42-Gal4/UAS-TDP-43, <i>PABP</i>^KG02359 = 22, N = 110, p<0.01. D42-Gal4/UAS-TDP-43, <i>Rs1</i>^K09514 = 21, N = 30, p = 0.8. (<b>B</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies with the <i>Nup50</i>^kg09557 mutation. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 21, N = 110; D42-Gal4/UAS-TDP-43, <i>Nup50</i>^KG09557 = 26, N = 111; <i>p</i><0.0001. (<b>C</b>) Survival curves of D42-Gal4 control flies with <i>Nup50</i>^kg09557 mutation. Median survival (days) of: D42-Gal4/+ = 67.5, N = 110; D42-Gal4/<i>Nup50</i>^KG09557 = 75, N = 105, p<0.0001.</p

Effects of cell cycle gene mutations on D42-Gal4/UAS-TDP-43 fly lifespan.

<p>(<b>A</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies in <i>string</i> and <i>greatwall</i> mutant backgrounds. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 26, N = 118; D42-Gal4/UAS-TDP-43, <i>string</i>^EY12388 = 27, N = 106, p<0.0001; D42-Gal4/UAS-TDP-43, <i>greatwall</i>^EP515 = 23, N = 67, p<0.0001. (<b>B</b>) Effects of the <i>string</i>^EY12388 allele in males. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 23.5, N = 60; D42-Gal4/UAS-TDP-43, <i>string</i>^EY12388 = 23, N = 52, p = 0.1831. (C) Effects of the <i>string</i>^EY12388 allele in females. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 28, N = 58; D42-Gal4/UAS-TDP-43, <i>string</i>^EY12388 = 32, N = 54, p<0.0001. (<b>D</b>) Survival curves of D42-Gal4 control flies with the <i>string</i>^EY12388 mutant allele, divided by gender. Median survival (days) of males: D42-Gal4/+ = 58, N = 49; D42-Gal4/<i>string</i>^EY12388 = 53.5, N = 60, p = 0.08. Median survival (days) of females: D42-Gal4/+ = 63, N = 53; D42-Gal4/<i>string</i>^EY12388 = 60, N = 60, p = 0.43.</p

Genomic map of chromosomal deficiencies around the <i>Hey</i> locus.

<p>Deficiency lines used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057214#pone-0057214-g008" target="_blank">Figure 8</a> and the location of genes tested in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057214#pone-0057214-g009" target="_blank">Figure 9</a> were as indicated. White boxes represented DrosDel collection while black boxes, Exelixis collection. Genomic mapping was adapted from Flybase GBrowse program (<a href="http://flybase.org/cgi-bin/gbrowse/dmel/" target="_blank">http://flybase.org/cgi-bin/gbrowse/dmel/</a>).</p

Validation of gene expression changes in TDP-43 flies.

<p>qPCR analysis of selected genes identified via microarray. (A) Upregulation of notch signaling genes by TDP-43 overexpression in the fly compound eye. (B) Changes of other genes by TDP-43 overexpression. GMR-Gal4/+ was treated as control sample. Data were presented as mean of four independent biological replicates with ±SEM.</p

Gene expression changes in inducible TDP-43 flies.

<p>All annotated genes identified by microarray comparison of ELAV-GS-Gal4/UAS-TDP-43 flies treated with RU-486 versus DMSO control are shown, using criteria of >2 fold change and p<0.05. Functions are from FlyBase, and include both experimentally derived functions and putative functions based on structure or homology.</p

Binding of TDP-43 to Hey mRNA and effects of Notch pathway mutations on D42-Gal4/UAS-TDP-43 fly lifespan.

<p>(<b>A</b>) Immunopurified HA-TDP-43 was incubated with increasing amounts of Hey pre-mRNA that was attached to the biotin-streptavidin beads. The captured HA-TDP-43 was eluted and examined by western-blot. HDAC6 was included as positive control for the assay. (<b>B</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies crossed to the mutant allele <i>Tom</i>⁹⁹ and <i>BobA</i>^f03024. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 26, N = 118; D42-Gal4/UAS-TDP-43, <i>Tom</i>⁹⁹ = 24, N = 102, p<0.05; D42-Gal4/UAS-TDP-43, <i>BobA</i>^f03024 = 26, N = 119, p<0.05. (<b>C</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies crossed to the mutant allele and <i>Hey</i>^f06656. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 24, N = 213; D42-Gal4/UAS-TDP-43, <i>Hey</i>^f06656 = 23, N = 221, p = 0.0843. (<b>D</b>) Survival curves of D42-Gal4/UAS-TDP-43 flies crossed to overexpression line UAS-Numb. Median survival (days) of: D42-Gal4/UAS-TDP-43 = 26, N = 118; D42-Gal4/UAS-TDP-43, UAS-Numb = 23, N = 57, p<0.0001.</p

Expanded gene expression changes in inducible TDP-43 flies.

<p>Genes are from same experiment and analysis as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057214#pone-0057214-t003" target="_blank">Table 3</a>, with less stringent criteria to identify potentially important genes. nACR, nicotinic acetycholine receptor.</p