Additional file 7: of Inhibition of NF-κB in astrocytes is sufficient to delay neurodegeneration induced by proteotoxicity in neurons

2018-09-11T05:00:00Z (GMT) by Y. Li O. Sibon P. Dijkers
a The UAS-Gal4 and the QUAS-QF system function independently. QUAS-QF-driven GFP expression specifically in neurons (GFP); UAS-Gal4-driven expression of myr-RFP specifically in astrocytes (RFP). Nuclei are stained with Hoechst (blue). b Genetic setup to inducibly express SCA3polyQ78 in neurons and simultaneously modulate gene expression in astrocytes. QUAS-QF was used to express SCA3polyQ78; UAS-Gal4 was used to modulate expression in astrocytes. Neuronally expressed QF2 (expressed under control of the pan-neuronal nSyb promoter) is suppressed by QS (expressed under control of the tubulin (tub) promoter). This suppression is alleviated by quinic acid, resulting in expression of SCA3polyQ78. For details on the fly lines used, see experimental procedures. c Quinic acid does not affect lifespan. Control flies were cultured on standard fly food with or without quinic acid, and the fraction of dead flies was determined over time. d Raising flies at 18 °C does not induce expression of Relish RNAi constructs. Progeny of flies ubiquitously expressing RNAi constructs targeting Relish (Relish RNAi #1 and Relish RNAi #2) raised at 18 °C were analyzed for expression of Relish as in Additional file 4b. e Effect of modulating expression of Relish in astrocytes on lifespan. The lifespan of control flies or flies expressing Relish RNAi or overexpression constructs specifically in astrocytes was analyzed. f Effect of modulating Relish expression in astrocytes on impairment of mobility induced by neuronal SCA3polyQ78 expression. Control flies or flies expressing inducible SCA3polyQ78 together with Relish RNAi or Relish overexpression constructs targeted to astrocytes were analyzed for mobility over time. Scoring was done as described in the experimental procedures. g Effect of modulating Relish expression in astrocytes on SCA3polyQ78 levels or aggregation. Head lysates of 15-day-old flies as in Fig. 5c were analyzed for expression of HA-tagged SCA3polyQ78 on western blot. Tubulin was used as a control for equal loading. h Expression levels of Abeta42. Two independent lines, QUAS-Abeta42 #5 and QUAS-Abeta42 #7 were analyzed for expression of Abeta42 using 6E10 antibody. Tubulin was used as a control for equal loading. Genotypes: a alrm-Gal4::UAS-myr-RFP; nSyb-QF2/QUAS-mCD8-GFP. c tub-QS/+; alrm-Gal4/+; nSyb-QF2::tub-QS/+. d Genotypes as in Additional file 4b. (e) -, tub-QS/+; alrm-Gal4/+; nSyb-QF2: tub-QS/+. Relish RNAi #1, Relish RNAi #2, or Relish overexpression, as in ‘-‘, but with UAS-Relish RNAi #1 or #2 or UAS-GFP-Relish. f Genotypes as in Fig. 5d. g As in e. h Control, nSyb-QF2/+. Abeta42 #5 or #7, QUAS-Abeta42/+; nSyb-QF2/+. (PDF 609 kb)