The pathogenic human Torsin A in Drosophila activates the unfolded protein response and increases susceptibility to oxidative stress

Abstract

Background: Dystonia1 (DYT1) dystonia is caused by a glutamic acid deletion (ΔE) mutation in the gene encoding Torsin A in humans (HTorA). To investigate the unknown molecular and cellular mechanisms underlying DYT1 dystonia, we performed an unbiased proteomic analysis.

Results: We found that the amount of proteins and transcripts of an Endoplasmic reticulum (ER) resident chaperone Heat shock protein cognate 3 (HSC3) and a mitochondria chaperone Heat Shock Protein 22 (HSP22) were significantly increased in the HTorA(ΔE)- expressing brains compared to the normal HTorA (HTorA(WT)) expressing brains. The physiological consequences included an increased susceptibility to oxidative and ER stress compared to normal HTorA(WT) flies. The alteration of transcripts of Inositol-requiring enzyme-1 (IRE1)-dependent spliced X box binding protein 1(Xbp1), several ER chaperones, a nucleotide exchange factor, Autophagy related protein 8b (ATG8b) and components of the ER associated degradation (ERAD) pathway and increased expression of the Xbp1-enhanced Green Fluorescence Protein (eGFP) in HTorA(ΔE) brains strongly indicated the activation of the unfolded protein response (UPR). In addition, perturbed expression of the UPR sensors and inducers in the HTorA(ΔE) Drosophila brains resulted in a significantly reduced life span of the flies. Furthermore, the types and quantities of proteins present in the anti-HSC3 positive microsomes in the HTorA(ΔE) brains were different from those of the HTorA(WT) brains.

Conclusion: Taken together, these data show that HTorA(ΔE) in Drosophila brains may activate the UPR and increase the expression of HSP22 to compensate for the toxic effects caused by HTorA(ΔE) in the brains.

Figure 1

A representative comparison of protein expression patterns between fly brains expressing HtorA^WT and HtorA^ΔE using 2-DE. (A) Differentially expressed protein spots of the HTorA^WT- and the HTorA^ΔE –expressing flies were labeled with Coomasie brilliant blue staining. Blue colors indicate lower amounts of proteins, and red colors represent significantly more proteins. (B) Representative 3D-views of 3 differentially expressed spots. (C) Quantification results of differentially expressed spots. * = p < 0.05, *** = p < 0.001.

Figure 2

The HTorA^ΔE-expressing flies had significantly increased HSC3 and HSP22 in their brains. (A and B) When the amounts of HSC3 were normalised with α-Tubulin as a loading control, the HTorA^ΔE-expressing brains had 1.4-fold more HSC3 than those of the HTorA^WT-expressing brains. However, Actin-enhanced Green fluorescence protein (Act-eGFP) expressing flies showed similar expression levels of HSC3. (C and D) In normal condition, HSP22 was detected from only the crude mitochondrial fraction of the HTorA^ΔE-expressing brains and it was 3.4-fold more abundant in the crude mitochondrial fraction of the HTorA^ΔE -expressing brains than that of the HTorA^WT-expressing brains before or following a heat shock. * = p < 0.05.

Figure 3

Transcripts of HSC3, HSP22, Tsf1 and IRE1-dependent spliced Xbp1 and Xbp1-eGFP signals were significantly increased in the HTorA^ΔE-expressing brains. Quantitative-RT-PCR results of genes encoding proteins displaying the largest alterations in expression in the 2-DE analysis. (A) Only the transcripts of heat shock protein cognate 3 (hsc3) but not those of hsc4 and hsc5 were significantly increased in the HTorA^ΔE flies. (B) The transcripts of transferrin1 (tsf1), heat shock protein 22 (hsp22), and sarcoplasimc calcium binding protein (scp1) were not changed. (C) The amount of the IRE1-dependent spliced form of Xbp1 mRNA (Xbp1 -23 bp) was increased in the HTorA^ΔE brains. (D) Compared with HTorA^WT, expression of HTorA^ΔE induced increased expression of Xbp1-eGFP in Drosophila heads. α-Tubulin was used as a loading control. (E) The normalised amount of the Xbp1-eGFP band in the HTorA^ΔE expressing brains was 3.68-fold higher than that in the HTorA^WT-expressing brains.

Figure 4

Increased sensitivities to oxidative and ER stressors and autophagy inhibitors in the HTorA^ΔE flies. (A) The hazard ratio (HR) of the HTorA^ΔE expressing flies reared on fly food containing 1% H₂O₂ was 2.073 times increased compared to those of the HTorA^WT expressing flies. (B) The HTorA^ΔE-expressing flies also showed 3.564 times increased HR to 20 mM paraquat induced oxidative stress. (C) When ER stress was induced using 12 mM tunicamycin, the HR of the HTorA^ΔE-expressing flies was 3.013 times increased compared with the HTorA^WT-expressing flies. (D-F) When a protein disulfide bond reducing compound DTT was applied with three different concentrations, including 5 mM (D), 25 mM (E), and 100 mM (F), there was no difference in HR between the HTorA^WT and the HTorA^ΔE-expressing flies. (G-I) The HTorA^ΔE flies were vulnerable to autophagy inhibitors, 10 mM 3-MA (G), 40 μM Wortmannin (H), and 40 μM LY294002 (I) compared with the HTorA^WT flies. HR = hazard ratio, 95% CI = 95% confident intervals of hazard ratio.

Figure 5

Transcripts of some ER stress sensors, chaperones, components of ERAD, and ATG8b were significantly altered in the HTorA^ΔE-expressing brains. The amount of ATF4, ATF6, calreticulin (A), ERp60, PDI, dGRP170, P58IPK (B), Hrd1, Hrd3, Derlin-1, EDEM1, EDEM2 (C) and ATG8b (D) transcripts in the HTorA^ΔE-expressing brains was significantly different from those of the HTorA^WT-expressing brains.

Figure 6

The distribution patterns of HSC3 in Optiprep density gradient factions acquired from the HTorA^ΔE-expressing brains were different from those of the HTorA^WT-expressing brains. (A) HSC3 was present in fraction Nos. 9–12 in the HTorA^WT-expressing brains, with the strongest signals in fraction No. 11. However, HSC3 was detected from fraction Nos. 8–12 in the HTorA^ΔE-expressing brains with the strongest signals found in fraction No. 10. (B ~ E) Proteins identified from fraction 9 (B), 10(C), 11(D), and 12(E) from the HTorA^ΔE-expressing brains were different from those form the HTorA^WT-expressing brains. (F) When all proteins identified from Faction 9 to 12 were pooled, fractions from HTorA^ΔE-expressing brains had 56 genes that were increased or unique, whereas 18 genes were increased or unique from the HTorA^WT-expressing brains. Gene ontology profiles for functions (F) and pathways (G) were displayed, respectively.

Figure 7

Down-regulation of hsc3, xbp1, ATF6 and Pek induced early death of the HTorA^ΔE flies. (A) When hsc3 expression was down-regulated by hsc-RNAi, it resulted in the induction of early death for the HTorA^ΔE flies. (B) Down-regulation of Xbp1 by mutant chromosome, xbp1 ^K13803 or xbp1 RNAi resulted in significantly earlier deaths among the HTorA^ΔE flies. (C) Decreased ATF6 in the HTorA^ΔE flies induced increased the HR of the HTorA^ΔE flies. (D) Down-regulated Pek in the HTorA^ΔE fly brains significantly increased the HR of the HTorA^ΔE flies.

Figure 8

The three axes of the UPR in Drosophila brains activated by HTorA^ΔE. HTorA^WT may not induce activation of the UPR in ER. However, HTorA^ΔE may induce activation of the three axes of the UPR signalling pathway by directly binding to HSC3 or by impairing protein trafficking and secretion from the ER that results in ER overload. The amounts of transcripts of the IRE1-dependent spliced Xbp1, AFT4, and ATF6 are consequently increased and initiate transcriptional up-regulation of ER-stress and UPR target genes, including HSC3, Xbp1, components of ERAD, ER chaperone, disulfide bond proteins, oxidative stress response proteins, and ATG8b in Drosophila brains. Consequently, the HTorA^ΔE flies showed an increased susceptibility to oxidative and ER stress and a prolonged UPR activation compared with the HTorA^WT flies.