Changes in Glutathione Redox Potential Are Linked to Aβ42-Induced Neurotoxicity  Zeenna A. Stapper, Thomas R. Jahn  Cell Reports  Volume 24, Issue 7, Pages 1696-1703 (August 2018) DOI: 10.1016/j.celrep.2018.07.052 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 24, 1696-1703DOI: (10.1016/j.celrep.2018.07.052) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 Establishment of In Vivo Models Combining Quantitative Redox Analysis with Drosophila Models of Aβ Aggregation (A and A′) Validation of the cyto-Grx1-roGFP2 redox sensor in neurons or glia cells in its fully oxidized (DA) and reduced state (DTT). (B) Dynamic ranges (DRs) of the biosensor. (C) Western blot analysis revealed no changes in soluble Aβ42 levels (left) and an accumulation of insoluble Aβ42 (right) over time (in days). (D) Longevity assays indicate a strong neurotoxicity in Aβ42 flies (black), while low neurotoxicity is observed in TAβ40 flies (pink) compared with control flies (blue) (n = 60–120 flies). (E) Immunohistochemical analysis of 12-day-old fly brains showed the strong deposition of ThS (green) positive amyloidogenic structures in Aβ42 flies, but almost none in TAβ40 flies. Total Aβ was stained with 6E10 (red). The first column shows a single-stack confocal microscopy image. All other images are maximum intensity projections (MIPs). Scale bars, 200 μm. Cell Reports 2018 24, 1696-1703DOI: (10.1016/j.celrep.2018.07.052) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Quantitative Redox Analysis Reveals Neuronal Glutathione Redox Changes (A–B′) Redox analysis using the glutathione redox sensor cyto-Grx1-roGFP2 (A and A′) and the H2O2 redox sensor cyto-roGFP2-Orp1 (B and B′) in neurons (A and B) and glia cells (A′ and B′) was performed in a time course (n = 3–5 fly brains per time point and genotype). The normalized cyto-Grx1-roGFP2 fluorescence intensity ratio increases in neurons, but not in glia cells, over time (A and A′). No changes in H2O2 levels are observed in neurons or in glia cells (B and B′). All error bars indicate SEM. ∗p ≤ 0,05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0.0001. Cell Reports 2018 24, 1696-1703DOI: (10.1016/j.celrep.2018.07.052) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 Activation of JNK Stress Response upon Aβ42 Amyloid Deposition (A and B) Analysis of JNK activation in fly brains using a stress-inducible promoter element fused to a fluorescent reporter (TRE-DsRed-2R) (B) over time (A) (n = 4 fly brains per time point and genotype). (C) Quantification. Depicted confocal microscopy images are MIPs. Scale bar, 200 μm. All error bars indicate SEM. ∗∗∗p ≤ 0.001. Cell Reports 2018 24, 1696-1703DOI: (10.1016/j.celrep.2018.07.052) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Glutathione Redox Imbalance Is Directly Linked to Aβ42-Mediated Neurotoxicity (A) Genetic manipulation of the GSH synthesis using the single expression system in 6-day-old Aβ42 flies had no impact on JNK activation. (B) Quantification of A (n = 15–18 fly brains per genotype). (C) Redox analysis of 12-day-old Aβ42 flies with Gclc-OE showed a further increase in the cyto-Grx1-roGFP2 fluorescent ratio. (D) Quantification of (C) (n = 15–17 fly brains per genotype). To DTT samples normalized 405 nm/488 nm ratios are shown. (E) Survival assays of Aβ42 flies with altered GSH synthesis revealed that changes in EGSH (C and D) of Aβ42 flies with Gclc-OE (purple) were associated with a further decreased lifespan, while Gclc-RNAi increased the lifespan of Aβ42 flies (green) (n ≥ 100 flies per genotype). (F and G) ELISA analysis of fly head extracts of 6-day-old flies revealed that manipulation of GSH synthesis did not change total (F) or insoluble (G) Aβ levels (n = 3 independent biological replicates). All error bars indicate SEM. ∗p ≤ 0.05, ∗∗∗p ≤ 0.001, and ∗∗∗∗p ≤ 0,0001. Depicted confocal microscopy images are MIPs. Scale bar, 200 μm. Cell Reports 2018 24, 1696-1703DOI: (10.1016/j.celrep.2018.07.052) Copyright © 2018 The Author(s) Terms and Conditions