Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation

Abstract

A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ∼1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-β and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3β is strongly upregulated due to TDP-43 expression, and reduced GSK-3β dosage is also a common suppressor of Aβ42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation.

Figure 1.

Genetic modifiers of tau-induced neurotoxicity identified from P lethal LOF screen. ‘Control’: w¹¹¹⁸/+;gl-tau/+. All other panels, except wild-type, contain one copy of gl-tau transgene in trans to one disrupted copy of the gene listed in the panel. Genes are listed alphabetically; non-annotated genes comprise the bottom row. Arrows point to necrotic plaques that were initially identified by light microscopy.

Figure 2.

Modifiers of tau-induced neurotoxicity show little effect on apoptosis. The 37 modifiers identified from P lethal and EY functional screens were tested for their ability to suppress the effects of the proapoptotic gene hid. P lethal control: w¹¹¹⁸;GMR-hid/+. All other P lethal genotypes have one copy of GMR-hid in trans to one disrupted copy of the listed gene. EY control: GMR-GAL4/w¹¹¹⁸;GMR-hid/+. All other EY genotypes have one copy of GMR-GAL4 on the first chromosome and one copy of GMR-hid in trans to the affected listed gene. Genes are listed alphabetically. Nearly all modifiers failed to show an effect on apoptosis when compared with controls. CamKI and Hr39 showed a moderate suppression of apoptosis but enhanced tau toxicity. Modifiers oho23B, Tango5 and Tis11 showed mildly increased phenotypes when compared with hid.

Figure 3.

Genetic modifiers of tau-induced neurotoxicity identified from EY collection screen. ‘Control’: GMR-GAL4/+;gl-tau/+. All other panels contain one copy of GMR-GAL4 on the X chromosome and one copy of gl-tau transgene in trans to the gene listed in the panel affected by the EY element. Genes are listed alphabetically.

Figure 4.

Quantification of eye volumes of modifiers. (A) Three-dimensional reconstructions of representative eyes of wild-type, control gl-tau (+/gl-tau), enhancer (Hop) and suppressor (shaggy) phenotypes. (B) Estimated eye volumes of modifier phenotypes from P lethal and EY screens indicated in scatter plots. Blue, suppressors; red, enhancers; black, control; green, wild-type. Black horizontal lines delineate range of control eye volumes; suppressors have larger eye volumes, whereas enhancers have smaller eye volumes when compared with control.

Figure 5.

Computational tau toxicity modifier network can predict novel tau modifiers. (A) Computational predictions of tau interactors based on high association with hits from the functional genetic screen. Hits from the functional screen are marked with asterisks. Size of the nodes is proportional to degree of connectivity with other genes in the network. Although all genes selected for inclusion have high connectivity to many genes in the network (see Materials and Methods for parameters of inclusion), for clarity only the strongest interactions with a quantified interaction (P-value <0.001) are indicated in blue lines. Each functional group is coded with a unique color; a node was color-divided to indicate whether multiple functions are attributed to that gene. (B) Network association genes—mitochondrial gene, Tom34, and RNA trafficking gene, csul—both strongly modify tau toxicity, showing strong suppression when overexpressed (GOF) and enhanced toxicity when expression is reduced (LOF). Tom34 and csul expression do not modify apoptosis (as assayed using the GMR-GAL4;GMR-hid phenotype), nor show any intrinsic eye phenotypes (as assayed with GMR-GAL4 alone). However, both show effects on the GMR-GAL4::UAS-Q108 phenotype—Tom34 suppresses Q108 toxicity, whereas csul enhances toxicity.

Figure 6.

Modifying tau toxicity does not require altering tau phosphorylation state. (A) Immunoblots from tau toxicity modifiers probed for total tau (T46 or E178) and tau phosphorylation at S202/T205 (AT8). β-Tubulin or β-actin shown as loading control. P lethal control: w¹¹¹⁸/+;gl-tau/+. EY control: GMR-GAL4/+;gl-tau/+. Genes labeled in red designate enhancers, whereas genes labeled in blue are suppressors. Blots for EY hits were visualized by two-color immunoblots with fluorescent secondaries and imaged with the Odyssey Near-IR Scanner (Li-Cor), but are shown in grayscale. Two-color immunoblots allowed for visualization and measurement of total tau (E178, rabbit IgG) and AT8 (mouse IgG) simultaneously. (B) Quantification of phosphorylated tau. Modifiers did not significantly alter total tau expression. AT8 levels were normalized to total tau; significant differences (P < 0.05) when compared with control are marked with asterisks. Approximately half (18 of 40) of the modifiers showed no significant differences in phospho-tau when compared with control. The other half (22 of 40) showed a significant difference in AT8; however, no pattern of phosphorylation correlated with enhancement or suppression of the tau phenotype. (C) Scatterplot of the mean values for AT8 signal of the 40 modifiers identified shows little difference in phosphorylation state between enhancers and suppressors.

Figure 7.

p38 interacts synergistically with tau to regulate ERK activity and ksr decreases GSK-3β and ERK activities. (A) Immunoblots and (B) quantification of kinase activity induced by Mekk1 overexpression, which enhanced tau toxicity. Mekk1 overexpression induced a modest significant increase in p38 activity (phospho-p38) independent of human tau expression. ERK activity (phospho-ERK) was equivalent for tau expression alone (first lane) and Mekk1 expression alone (third lane); however, tau and Mekk1 co-overexpression strongly reduced phospho-ERK levels (second lane). Overexpression of Mekk1 also strongly reduced S202/T205 tau phosphorylation levels (AT8), but did not alter total tau levels (T46). No significant difference was observed for GSK-3β activity (phospho-GSK-3β-Ser9). (C) Both enhancer bancal and suppressor ksr decrease ERK activity, but ksr also strongly reduced GSK-3β activity, while bancal trended to increase GSK-3β activity.

Figure 8.

GSK-3β/shaggy is a common suppressor of S11A, Aβ42 and TDP-43 toxicity, whereas S2A is resistant to all enhancers. (A) Expression of the phosphorylation-resistant Tau^S11A isoform shows strong toxicity, stronger than Tau^WT expression. However, select suppressors of gl-Tau^WT were also able to suppress S11A toxicity. (B) Expression of the phosphorylation-resistant Tau^S2A isoforms shows no toxicity. All enhancers identified in the screen were tested against S2A phenotype; five representative enhancers are depicted. No enhancer could induce a rough eye phenotype with Tau^S2A. Western blots confirm robust expression of Tau^S2A protein with all enhancers assayed. (C) NC2α and SdhB, which suppressed both tau and polyglutamine toxicity, were tested for their effects on Aβ42 and TDP-43^Q331K toxicity. Neither gene showed any significant effect on the GMR-GAL4::UAS-Aβ42 phenotype, nor on the GMR-GAL4::UAS-hTDP-43^Q331K phenotype when compared with controls. However, shaggy/GSK-3β showed suppression of toxicity, as indicated by increased size and eye volume of the Aβ42 eye, and by increased color and pigment retention in the TDP-43 eye. Modifier ksr shows a mild suppression of Aβ42 and TDP-43 toxicity, although not as robust as sgg suppression. Scale bar, 100 μm. (D) Expression of hTDP-43^Q331K shows a robust increase in GSK-3β activity, as indicated by western blot of reduced Ser9 phosphorylation of GSK-3β (P = 0.004).

Figure 9.

Modifiers of tau-induced neurotoxicity do not modify polyglutamine toxicity. The 37 modifiers identified from P lethal and EY screens were crossed to w¹¹¹⁸;GMR-GAL4, UAS-Q108/CyO. Controls: w¹¹¹⁸;GMR-GAL4, UAS-Q108/+. All other genotypes have one copy of GMR-GAL4, UAS-Q108. The gene listed refers to the allele affected by P element or EY element; genes are listed alphabetically. Most tau modifiers showed no effect on polyglutamine toxicity; only NC2α and SdhB showed suppression.