Proteasome dysfunction in Drosophila signals to an Nrf2-dependent regulatory circuit aiming to restore proteostasis and prevent premature aging

Abstract

The ubiquitin-proteasome system is central to the regulation of cellular proteostasis. Nevertheless, the impact of in vivo proteasome dysfunction on the proteostasis networks and the aging processes remains poorly understood. We found that RNAi-mediated knockdown of 20S proteasome subunits in Drosophila melanogaster resulted in larval lethality. We therefore studied the molecular effects of proteasome dysfunction in adult flies by developing a model of dose-dependent pharmacological proteasome inhibition. Impaired proteasome function promoted several 'old-age' phenotypes and markedly reduced flies' lifespan. In young somatic tissues and in gonads of all ages, loss of proteasome activity induced higher expression levels and assembly rates of proteasome subunits. Proteasome dysfunction was signaled to the proteostasis network by reactive oxygen species that originated from malfunctioning mitochondria and triggered an Nrf2-dependent upregulation of the proteasome subunits. RNAi-mediated Nrf2 knockdown reduced proteasome activities, flies' resistance to stress, as well as longevity. Conversely, inducible activation of Nrf2 in transgenic flies upregulated basal proteasome expression and activity independently of age and conferred resistance to proteotoxic stress. Interestingly, prolonged Nrf2 overexpression reduced longevity, indicating that excessive activation of the proteostasis pathways can be detrimental. Our in vivo studies add new knowledge on the proteotoxic stress-related regulation of the proteostasis networks in higher metazoans. Proteasome dysfunction triggers the activation of an Nrf2-dependent tissue- and age-specific regulatory circuit aiming to adjust the cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis circuit accelerates aging.

Keywords: Aging; Drosophila; Keap1; Nrf2; proteasome; somatic tissue.

© 2013 The Anatomical Society and John Wiley & Sons Ltd.

Figures

Fig. 1

Sustained proteasome dysfunction in young somatic tissues after eclosion disrupts proteostasis, reduces locomotor performance, and shortens flies lifespan in a dose-dependent manner. (A) Relative (%) 26S (A1) and 20S (A2) proteasome activities in Drosophila somatic tissues following exposure for 6–9 days of young flies to the indicated concentrations of PS-341. (B, C) Immunoblot analyses of total protein ubiquitination (Ub) (B) and carbonylation (DNP) (C) in somatic tissues of young flies treated [as in (A)] with increasing concentrations of PS-341. (D) Climbing activity (%) at the indicated ages of either not treated flies (normal aging) or flies exposed to the shown doses of PS-341. (E) Longevity curves of flies exposed to the indicated concentrations of PS-341. Flies median lifespan and comparative statistics are reported in Table S1 (Supporting information). (F) Immunoblot analyses of AGEs-modified proteins in somatic tissues of flies treated (or not) with PS-341 for 17 days. In (B), (C), and (F), probing with GAPDH was used as a loading reference. Controls in (A) were set to 100%. Dashed circles in (D) indicate not significant differences vs. the control (normal aging). Bars, ± SD (n = 3). *P < 0.05; **P < 0.01.

Fig. 2

Proteasome inhibition in young somatic tissues results in dose-dependent upregulation of the proteasome genes and protein subunits, higher proteasome assembly rates, and reactive oxygen species (ROS) accumulation. (A) Representative immunoblot analyses of young female and male somatic tissues protein samples probed with antibodies against Rpn7, 20S-α, and β5 after exposure of flies to the indicated doses of PS-341. (B) RT–PCR analyses of the rpn11, α7, β1, β2, β5 (B1), atg6, atg8, cncC, and keap1 (B2) genes expression following proteasome inhibition. (C) Co-immunoprecipitation of the 20S-α and β5 proteins (indicating assembled proteasome) in lysates from young Drosophila somatic tissues after treatment of flies with PS-341. Immunoprecipitation (IP) was performed with an anti-20S-α antibody, and immunoprecipitates were probed (IB) with anti-20S-α, anti-β5, and anti-Ub antibodies. (D) Comparative immunoblot analyses of young (♀/♂) and aged (♀/♂ aged) somatic tissues protein samples probed with antibodies against Rpn7, 20S-α, β5, and Ub; flies were exposed to 5–20 µm of PS-341. (E) Relative (%) ROS (E1, E3) and H2O2 (E2) levels in the somatic tissues of young (E1, E2) and old (E3) flies exposed to PS-341 (control samples were set to 100%); in absolute values, aged flies had higher endogenous ROS and H2O2 levels (see also, Fig. S2B). In all cases, flies were exposed to PS-341 for 4 days. GAPDH probing (A), (D) and rp49 gene (B) expression were used as reference for total protein and RNA input, respectively; Coommassie Blue (CB) stain of unbound fraction in (C) depicts total protein input. Bars, ± SD (n = 2); *P < 0.05.

Fig. 3

Proteasome impaired function-related reactive oxygen species (ROS) start accumulating in young somatic tissues after 2–4 days of treatment and correlate with proteasome protein subunits upregulation. (A, B) Relative fold change of 26S (A1) or 20S (A2) proteasome activities and ROS levels (B), in time course experiments following administration of 1 µm PS-341 to flies; controls were set to 1. Although proteasome is already inhibited at day 1, ROS burst after ~2–4 days of treatment. (C) Representative immunoblot analyses of young somatic tissues protein samples probed with antibodies against Rpn7, 20S-α, β5, and Ub after treatment of flies as in (A). (D) Representative immunoblot analyses of somatic tissues protein samples derived from young flies treated (or not) for 3 days with 1 µm PS-341 or with 1 µm PS-341 and the indicated doses of Tiron; blots were probed with antibodies against Rpn7, 20S-α, β5, and Ub. GAPDH was used as a reference. Bars, ± SD (n = 2). *P < 0.05 [in (A), values lower than 0.8 were significant]; **P < 0.01.

Fig. 4

Proteasome dysfunction in young somatic tissues disrupts mitochondria function triggering increased production of reactive oxygen species (ROS). (A) Immunoblotting analysis of total protein carbonylation (left panel) and ubiquitinated proteins (right panel) in isolated mitochondria from young flies’ somatic tissues following exposure to PS-341 for 12 days. Probing with an antibody against OxPhos complex IV and Ponceau staining (PN) were used as protein-loading reference. (B) Relative (%) succinate dehydrogenase activity at day-13 (B1) and respiratory chain complex II/III activity (B2) at time course experiments following PS-341 administration to the flies. (C, D) Relative% ROS levels in isolated tissue mitochondria [(C); exposure to PS-341 for 1 up to 17 days] and in whole cell lysates, mitochondria, and cytosolic purified fractions [(D); flies were treated with the inhibitor for 3–14 days]. (E) ROS-related fluorescence units per protein µg (left panel) or relative (%) (right panel) ROS levels in isolated mitochondria from somatic tissues of young (Y) or old (O) flies treated for 3–4 days with PS-341. In all experiments, flies were exposed to 1 µm PS-341. Bars, ± SD (n = 3); *P < 0.05. Controls (%) in (B–E) were set to 100%.

Fig. 5

Proteasome loss of function triggers activation of antioxidant response elements (AREs) in young Drosophila somatic tissues. (A1) Relative (%)green fluorescent protein (GFP) fluorescent levels following proteasome inhibition in young transgenic gstD-ARE:GFP, gstD-mARE:GFP, and 4XARE:GFP flies. (A2) Direct viewing of proteasome inhibition–related AREs activation by whole-body GFP fluorescence detection at Confocal Laser Scanning Microscope (CLSM). (B) Relative (%) GFP fluorescent levels following proteasome inhibition in aged transgenic gstD-ARE: GFP and 4XARE:GFP flies. (C) GFP-related fluorescence levels per somatic tissue protein µg (C1) or (%) (C2) following PS-341-mediated proteasome inhibition in young (Y) or old (O) transgenic gstD-ARE:GFP or gstD-mARE:GFP flies. In all cases, flies were exposed to the indicated concentrations of PS-341 for 4 days. Controls in (A1), (B) and (C2) were set to 100%. Bars, ± SD (n = 2). *P < 0.05; **P < 0.01.

Fig. 6

RNAi-mediated CncC knockdown in eclosed flies suppresses proteasome activities, disrupts proteostasis, and decreases resistance to proteotoxic stress; it also reduces flies’ lifespan and abolishes proteasome components upregulation after proteasome loss of function. (A) RT–PCR of the cncC, keap1 (A1) and rpn11, α7, β5, gstD1, and atg8 (A2) genes expression levels in somatic tissues samples following RNAi-mediated CncC knockdown. rp49 gene expression was used as reference; to induce transgene expression, flies were fed with RU486 for 4 days. (B) Relative (%) 26S proteasome activity in flies somatic tissues after CncC knockdown. (C) Immunoblotting analysis of total ubiquitinated (Ub) protein levels in lysates of transgenic flies’ somatic tissues after CncC knockdown (flies were fed with RU486 for 14 days); CB stain was used as a reference of total protein input. (D) Survival rates of flies expressing the cncCRNAi transgene after exposure (or not) to PS-341 for 6 days; RU486 was administered (or not) for the whole duration of the experiment. (E) Longevity curves of transgenic flies grown in the absence [CncC RNAI(−)] or in the continuous presence [CncC RNAI(+)] of RU486. Flies’ median lifespan and comparative statistics are reported in Table S1 (Supporting information). (F) Representative immunoblot analyses of young somatic tissues protein samples probed with antibodies against Rpn7, 20S-α, β5, and Ub. Transgenic flies were treated (or not) with RU486 and the indicated concentrations of PS-341 for 4 days. As it is evident, CncC RNAi abolishes proteasome subunits induction after proteasome loss of function. GAPDH was used as reference. Bars, ± SD (n = 2). *P < 0.05; **P < 0.01. Controls in (B) and (D) were set to 100%.