Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention

Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention

Parkin’ control: regulation of PGC-1α through PARIS in Parkinson’s disease

Summary and comment on a recent Cell paper entitled ‘PARIS (ZNF746) repression of PGC-1α contributes to neurodegeneration in Parkinson’s disease’ (Shin et al., 2011)

From white to brown fat through the PGC-1α-dependent myokine irisin: implications for diabetes and obesity

Summary and comment on a recent Nature paper entitled ‘A PGC1-α-dependent myokine that drives brown-fat-like development of white fat and thermogenesis’ (Boström et al., 2012)

Fine-tuning autophagy maximises lifespan and is associated with changes in mitochondrial gene expression in Drosophila.

Increased cellular degradation by autophagy is a feature of many interventions that delay ageing. We report here that increased autophagy is necessary for reduced insulin-like signalling (IIS) to extend lifespan in Drosophila and is sufficient on its own to increase lifespan. We first established that the well-characterised lifespan extension associated with deletion of the insulin receptor substrate chico was completely abrogated by downregulation of the essential autophagy gene Atg5. We next directly induced autophagy by over-expressing the major autophagy kinase Atg1 and found that a mild increase in autophagy extended lifespan. Interestingly, strong Atg1 up-regulation was detrimental to lifespan. Transcriptomic and metabolomic approaches identified specific signatures mediated by varying levels of autophagy in flies. Transcriptional upregulation of mitochondrial-related genes was the signature most specifically associated with mild Atg1 upregulation and extended lifespan, whereas short-lived flies, possessing strong Atg1 overexpression, showed reduced mitochondrial metabolism and up-regulated immune system pathways. Increased proteasomal activity and reduced triacylglycerol levels were features shared by both moderate and high Atg1 overexpression conditions. These contrasting effects of autophagy on ageing and differential metabolic profiles highlight the importance of fine-tuning autophagy levels to achieve optimal healthspan and disease prevention

Aβ42 inhibits cncC activity in <i>Drosophila</i>.

<p>(A) Nrf2/cncC activity was measured by crossing <i>gstD1</i>(ARE)–GFP reporter flies to UAS-attP Aβ-expressing lines, under control of the <i>Drosophila</i> necrotic signal peptide [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref036" target="_blank">36</a>], then measuring GFP expression in heads by Western blotting. (B) Quantitation of WB depicted in (A) above. A separate experiment was run for each Aβ-expressing line. GFP expression was normalized to actin, for–RU and +RU samples, then expressed as a percentage of the average–RU value for each blot to enable comparison. ArcAβ42 significantly reduced GFP expression compared to controls (** <i>p</i>< 0.01 comparing +RU to–RU). <i>p</i>>0.05 comparing +RU to–RU for WT Aβ40 and 42 lines (student’s t-test). Data are presented as means ± SEM and were analysed by student’s t-test for each genotype. N = 4 biological repeats of 10 fly heads per sample. (C) Analysis of Nrf2/cncC activity in response to high levels of WT Aβ42 expression. <i>gstD1</i>(ARE)–GFP reporter flies were crossed to flies expressing a tandem double copy of WT Aβ42 under the control of the argos signal peptide [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref037" target="_blank">37</a>]. Data were analysed as described in (B). * <i>p</i><0.05 comparing -RU to +RU (student’s t-test). N = 4 biological repeats of 10 fly heads per sample. (D) Heatmaps depicting gene ontology (GO) categories altered by ArcAβ42 expression in + RU vs–RU UAS-Arc Aβ42>elavGS flies (column Arc Aβ42) and that overlap with categories altered by cncC over-expression[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref038" target="_blank">38</a>]; column cncC). Red indicates higher expression; blue indicates lower expression (scale = log₁₀ fold change). A random insertion UAS-ArcAβ42 line was used for these experiments[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref039" target="_blank">39</a>]. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.s001" target="_blank">S1A Fig</a> for effects of ArcAβ42 induction of Nrf2 activity in heads and bodies using these flies. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.s001" target="_blank">S1B Fig</a> for Venn diagrams of reciprocal effects of ArcAβ42 and cncC activation on GO category expression.</p

Keap1 inhibition and protein degradation pathways in Aβ42 flies.

<p>(A) Keap1 did not modify autophagy in Aβ42-expressing flies, as measured by the ratio of ATG8-II to ATG8-I levels. Data are presented as means ± SEM. <i>P</i>>0.05 (one-way ANOVA and Tukey’s post-hoc analysis). N = 4 biological repeats of 10 fly heads per condition. (B) Proteasome activity, as measured using the fluorogenic peptide substrate LLVY-AMC, was increased by Keap1 modification in Aβ42-expressing flies. Data are presented as mean activities (pmoles/min/mg protein) ± SEM. <i>P</i><0.05 comparing–RU, +RU and–RU, Keap1 del to +RU, Keap1 del flies (one-way ANOVA and Tukey’s HSD). N = 4–5 repeats of 10 fly heads per condition.</p

Keap1 and GSK-3 in the regulation of Nrf2 in Alzheimer’s disease.

<p>(A) Aβ42 peptide inhibits activity of Nrf2, and this may explain the increased presence of xenobiotic and oxidative stress markers observed in Alzheimer’s disease. (B) Although lithium can activate Nrf2 at high concentrations, its protective effect against Aβ42 toxicity appears to be mainly Nrf2-independent, reducing Aβ42 levels by inhibiting translation[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref047" target="_blank">47</a>] and preventing oxidative damage. More specific GSK-3 inhibitors are required to confirm the precise role of GSK-3 in rescuing Nrf2 deficits in neurodegenerative disease. (C) Genetic and pharmacological inhibition of Keap1 can rescue Aβ42-induced Nrf2 inhibition and neuronal toxicity by preventing xenobiotic damage and activating degradation of Aβ42 peptide. (D) Keap1 inhibitors may serve as effective therapies for AD and, in combination with GSK-3 inhibitors, may provide added benefits in preventing neurodegeneration through non-overlapping mechanisms.</p

Direct Keap1-Nrf2 inhibitors protect mouse neurons from Aβ toxicity.

<p>(A) Molecular structure of compound 22h (1-(3-methylphenyl)-4-(3-nitrophenyl)-1,2,3-triazole[<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.ref022" target="_blank">22</a>]. (B) Aβ oligomer toxicity, as measured by sensitivity to resazurin (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#sec014" target="_blank">methods</a>), in SH-SY5Y cells treated with a 50% dilution of either WT or 7PA2 CHO cell conditioned medium (** <i>p</i><0.01 comparing DMSO, wtCM to DMSO, 7PA2CM). An Nrf2 activator (CDDO-Me), the Keap1-Nrf2 disruptor (22h) and the GSK-3 inhibitor (TDZD8) were administered, at their EC₅₀ concentrations for Nrf2 activation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#pgen.1006593.s007" target="_blank">S7 Fig</a>), and their effects on resazurin sensitivity measured in both WT and 7PA2-treated cells. At 1 μM, TDZD8 was toxic to cells under both treatment conditions (*** <i>p</i><0.001 comparing TDZD8, wtCM and TDZD8, 7PA2CM to DMSO, wtCM). CDDO-Me and 22h exerted no toxicity at their effective concentrations in wtCM-treated cells (<i>P</i>>0.05). CDDO-Me slightly, but non-significantly, protected against Aβ oligomer toxicity (<i>P</i>>0.05 comparing DMSO, 7PA2CM to 10 nM CDDO, 7PA2CM cells) but 22h significantly protected against Aβ-induced damage (** <i>p</i><0.01 comparing DMSO, 7PA2CM to 10 μM 22h, 7PA2CM). Data are presented as mean fluorescence units (as a ratio of DMSO, wtCM-treated cells) ± SEM and were analysed by two-way ANOVA and Tukey’s HSD post-hoc comparisons. N = 8 wells per condition. (C) Representative confocal images of mouse cortical neurons treated with Tg2576CM in the presence or absence of the Keap1-Nrf2 inhibitor, 22h. Scale bar 5μm. (D) Tg2576CM reduced total spine density of mouse cortical neurons (*<i>p</i><0.05 comparing Tg2576CM, 0.01% DMSO to wtCM, 0.01% DMSO) and this was rescued by treatment with 22h in a dose-dependent manner (*<i>p</i><0.05 comparing Tg2576CM, 0.01% DMSO to Tg2576CM, 10 μM 22h; <i>p</i>>0.05 comparing Tg2576CM, 1 μM 22h to Tg2576CM, 0.01% DMSO and Tg2576CM, 10 μM 22h). 22h did not exert cytotoxicity (<i>p</i>>0.05 comparing wtCM, 0.01% DMSO to wtCM, 10 μM 22h). Data are representative of means ± SEM and were analysed by one-way ANOVA and Tukey’s post-hoc analyses. N = 6–17 cells, from 2 independent wells, per condition. (E) Aβ42 peptide levels in conditioned media following 16h pre-treatment plus 24h CM treatment of WT mouse cortical neurons with or without 22h. (<i>p</i>>0.05 comparing 10 μM 22h to 0.01% DMSO for both wtCM and Tg2576CM). (F) mRNA expression of Nrf2 target genes, measured by qPCR, using extracts from primary cortical neurons from (E) above. Comparing 10 μM 22h to 0.01% DMSO, <i>p</i> = 0.008 (Hmox1), 0.013 (Srnx1) and 0.008 (xCT) (Wilcoxon rank sign test). N = 5–6 wells, per condition. 22h and DMSO data represent pooled means ± SEM from both wtCM and Tg2576CM-treated cells.</p

Keap1 inhibition enhances degradation of the Aβ42 peptide.

<p>(A) Lowering Keap1 reduced total Aβ42 peptide levels in 14 day-old flies. Data are presented as means ± SEM. <i>P</i><0.05 comparing +RU or +RU, UAS-Keap1 to +RU, Keap1 del or +RU, Keap1 EY5 flies (one-way ANOVA and Tukey’s HSD). N = 4 biological repeats of 5 fly heads per sample. (B) Aβ42 mRNA in 14-day-old Keap1 del flies. Data are presented as means ± SEM. <i>P</i><0.05 comparing +RU to +RU, Keap1 del flies (one-way ANOVA and Tukey’s HSD). N = 4 replicates of 20 flies per condition. (C) Reducing Keap1 lowered Aβ42 peptide levels, compared to controls. Data are presented as means ± SEM. <i>P</i><0.05 comparing +RU to +RU, Keap1 del following 14 days of induction. No significant difference was observed between genotypes at all other time-points (two-way ANOVA and Tukey’s HSD). N = 3–4 replicates of 5 fly heads per condition. (D) Inhibition of Keap1 led to the degradation of Aβ42 peptide following removal of the inducer RU486. Data are presented as means ± SEM. <i>P</i><0.05 comparing +RU 7d, Keap1 del flies at 0 to 3, 7 and 14 days following switch-off. No significant difference was observed between time-points following switch-off for +RU 7d control flies (two-way ANOVA and Tukey’s HSD). N = 4 repeats of 5 fly heads per condition. (E) Analysis of insoluble Aβ42 peptide (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006593#sec014" target="_blank">methods</a>) in 17-day-old Keap1 LOF flies following 10 days RU486 induction and 7 days switch-off. <i>P</i><0.05 comparing +RU, to +RU, Keap1 del or +RU, Keap1 EY5 flies (one-way ANOVA and Tukey’s HSD). N = 3–4 biological repeats of 5 fly heads per sample.</p