Differential regulation of proteasome functionality in reproductive vs. somatic tissues of Drosophila during aging or oxidative stress

Abstract

Proteasome is central to proteostasis maintenance, as it degrades both normal and damaged proteins. Herein, we undertook a detailed analysis of proteasome regulation in the in vivo setting of Drosophila melanogaster. We report that a major hallmark of somatic tissues of aging flies is the gradual accumulation of ubiquitinated and carbonylated proteins; these effects correlated with a ~50% reduction of proteasome expression and catalytic activities. In contrast, gonads of aging flies were relatively free of proteome oxidative damage and maintained substantial proteasome expression levels and highly active proteasomes. Moreover, gonads of young flies were found to possess more abundant and more active proteasomes than somatic tissues. Exposure of flies to oxidants induced higher proteasome activities specifically in the gonads, which were, independently of age, more resistant than soma to oxidative challenge and, as analyses in reporter transgenic flies showed, retained functional antioxidant responses. Finally, inducible Nrf2 activation in transgenic flies promoted youthful proteasome expression levels in the aged soma, suggesting that age-dependent Nrf2 dysfunction is causative of decreasing somatic proteasome expression during aging. The higher investment in proteostasis maintenance in the gonads plausibly facilitates proteome stability across generations; it also provides evidence in support of the trade-off theories of aging.

Keywords: Keap1; Nrf2; antioxidant response elements; gonads.

Figures

Figure 1.

Drosophila somatic tissue aging is characterized by accumulation of ROS, H2O2, and oxidized (carbonylated) and AGE-modified proteins, as well as by activation of ARE-mediated transcription and a significant decline of proteasome peptidase activity. A1–B3) Relative percentage of ROS (A1) and H2O2 (A2), protein carbonylation (B1) and quantitative analysis (B2), and AGE-modified protein levels (B3) in somatic tissues of young (Y), middle-aged (M), and old (O) female or male flies. DNP, 1,3-dinitrophenylhydrazone. C) Relative percentage of GFP fluorescence during aging of female and male gstD-ARE:GFP (gstD-ARE:GFP/II), 4XARE:GFP (4XARE:GFP-16(R7)/II), and gstD-mARE:GFP (gstD-mARE:GFP/III) transgenic flies; as is evident, reporter induction required a functional ARE. Median life spans of transgenic flies were as follows: 4XARE:GFP, 41±1.45 d; gstD-ARE:GFP, 39±9.29 d; gstD-mARE:GFP, 52±2.87 d. D1, D2) Comparative analyses of somatic tissue proteasome activity in single female or male fly preparations or after pooling female or male flies. D1) Fluorescence units per microgram of tissue protein following measurement of the 26S proteasome peptidase activity CT-L (LLVY/β5) in single female or male fly preparations; mean value in female flies was 6.195 ± 0.81 U/μg, whereas in males, it was 5.162 ± 0.68 U/μg. D2) Relative activity (%) of 26S CT-L (LLVY/β5) and C-L (LLE/β1) in preparations of pooled (10–12 flies) female or male flies. E, F) Relative activity (%) of 26S (E) and 20S (F) proteasome peptidase in somatic tissues during aging; sex-independent decline of activities is evident. Actin or GAPDH probing in B1, B3 was used as reference for equal protein loading. Bars represent means ± sd; n = 2 (B1–B3, C, D1); n = 3 (A1, A2, D2, E, F). *P < 0.05; **P < 0.01.

Figure 2.

Aging of Drosophila somatic tissues is characterized by lower proteasome expression and assembly rates. A1, A2) Representative immunoblot (A1) and densitometric analysis (A2) of female and male somatic tissue protein samples probed with antibodies against Rpn7, 20S-α, β5, and ubiquitin. B) RT-PCR densitometric analyses of rpn11, α4, α7, β1, β2, and β5 mRNA expression levels during aging of somatic tissue. C) Immunoprecipitation of the 20S-α and β5 protein interaction (indicating assembled proteasome) in lysates from young and aged Drosophila somatic tissues. Lysates were immunoprecipitated (IP) with antibodies against 20S-α, and immunoprecipitates were probed by immunoblotting (IB) with anti-20S-α and anti-β5 antibodies. GAPDH protein probing was used as a reference for protein loading (A1) and rp49 gene expression as reference for total RNA input (B). Densitometric data indicate relative expression against a loading reference followed by normalization against controls, namely samples from young flies; n = 3 (A2); n = 2 (B). Bars represent means ± sd. *P < 0.05, **P < 0.01 vs. controls.

Figure 3.

Gonads of aged organisms retain low levels of carbonylated proteins and relatively stable 26S and 20S proteasome peptidase activity. A) Relative percentage of ROS in the ovaries (OV) and spermathecae (SP), showing higher ROS levels during aging. B) ARE-mediated transcription (quantified as percentage GFP fluorescence) in female and male gonads during aging of transgenic 4XARE:GFP and gstD-ARE:GFP flies. C1–C3) Immunoblotting analyses (C1) and quantitative analysis (C2) of total protein carbonylation and immunoblotting analysis of AGE-modified proteins (C3) in female and male gonads during aging; probing with GAPDH was used as protein loading reference. DNP, 1,3-dinitrophenylhydrazone. D, E) Relative activity (%) of 26S (D) and 20S (E) proteasomes in Drosophila ovaries and spermathecae during aging. Except for T-L activity, which was found to decline in the aged ovarian tissue, in all other cases, proteasome activity was either stable or increased (e.g., CT-L in SP). Bars represent means ± sd;n = 3 (A); n = 2 (B--E). *P < 0.05, **P < 0.01.

Figure 4.

Gonads from aged flies retain substantial proteasome expression levels and assembled proteasomes. A1, A2) Representative immunoblotting (A1) and densitometric (A2) analysis of ovary (OV) and spermatheca (SP) protein samples probed with antibodies against Rpn7, 20S-α, β5 and ubiquitin. Down-regulation of protein expression mostly refers to the 19S subunit Rpn7, and it is less pronounced than in somatic tissues (see Fig. 2A). Dashed line bracket indicates polypeptides showing increased ubiquitination in aged ovaries; arrows indicate polypeptides with similar ubiquitination levels; solid line bracket indicate polypeptides with reduced ubiquitination. B) RT-PCR densitometric analyses of the rpn11, α4, α7, β1, β2, and β5 mRNA expression levels in the gonads during aging. C) Immunoprecipitation of the 20S-α and β5 protein interaction (indicating assembled proteasomes) in lysates from young and aged Drosophila ovaries. Immunoprecipitation (IP) was performed with an anti-20S-α antibody, and immunoprecipitates were probed by immunoblotting (IB) with anti-20S-α and anti-β5 antibodies; Coomassie blue (C.B.) stain depicts total protein input. GAPDH protein probing was used as a reference for equal protein loading (A1) and rp49 gene expression as reference for total RNA input (B). Densitometric analysis (n=2) was performed as in Fig. 2. Bars represent means ± sd. *P < 0.05.

Figure 5.

Gonads of young flies possess more abundant and more active proteasomes than somatic tissues. A1–A5) Representative immunohistochemical staining of whole female fly sections with an anti-20S-α proteasome subunit antibody. The antigen was distributed in head (H), thorax (T), and abdomen (A) tissues (A1). More intense staining was seen in the ovarian tissue (ov; A1, dashed line), in the neural tissues of the head [e.g., the eye (E) and the brain (B); A2], and in muscle (M; A3). In the ovarian follicles (F; A4), the 20S-α proteasome subunit was distributed in the nurse cells (NC), the oocyte (OC), and the follicular epithelium (FC) (A5). In all cell types, (including muscle cells; A3) both cytoplasmic and nuclear (arrows) staining was seen. Scale bars = 50 μm. B) Comparative RT-PCR analyses of mRNA expression levels of indicated proteasome subunits in somatic (SO) tissues and gonads (OV, ovary; SP, permathecae) of female and male flies. C) Immunoblotting analysis of proteasome subunit expression levels in gonads and somatic tissues of female and male flies. D) Immunoprecipitation (IP) of 20S-α and β5 proteasome subunit interaction (indicating assembled proteasomes) in lysates from Drosophila gonads and somatic tissues. Immunoprecipitation was performed with the anti-20S-α antibody, and immunoprecipitates were probed by immunoblotting (IB) with anti-20S-α and anti-β5 antibodies. E) Relative activity (%) of 26S (top panel) and 20S (bottom panel) proteasomes in somatic tissues and gonads of Drosophila. In nearly all cases, significantly higher proteasome peptidase activity was found in the gonads. F1, F2) Immunoprecipitation of the 20S-α proteasome subunit followed by immunoblotting analysis of 20S-α and β5 subunits. A minimal amount of the anti-20S-α antibody was used to achieve the immunoprecipitation of equal amounts of proteasomes from different tissues. F1) Indeed, although the unbound ovarian fraction contained (compared to somatic tissues) higher amounts of proteasomes, we detected similar amounts of assembled proteasomes in the eluate. F2) Relative activity (%) of 26S proteasome in the unbound fraction and immunoprecipitated proteasomes in the female soma and ovaries. Note higher endogenous proteasome catalytic activity in the immunoprecipitated ovarian proteasomes. All assays were done in 10- to 13-d-old flies. GAPDH and rp49 were used as references for equal total protein and RNA input, respectively. Bars represent means ± sd; n = 2. *P < 0.05, **P < 0.01.

Figure 6.

Gonad proteasome activity is more resistant to oxidative stress than that of somatic tissues and retains active antioxidant responses, in an age-independent manner. A1, A2) Immunoblot (A1) and RT-PCR (A2) analyses showing stable or increased protein (A1) and mRNA (A2) expression of proteasome subunits in somatic tissues of young (mixed population) flies exposed to 10 mM t-BHP for 8–13 d. B) Relative activity (%) of 26S proteasome in somatic tissues of young Drosophila following treatment with 10 mM t-BHP as in A or with 20 mM t-BHP for 3 or 4 d; in most cases, proteasome activity declined. C) Immunoblot analyses of the indicated proteasome protein subunits, showing stable or increased expression levels in ovaries (OV; C1) and spermathecae (SP; C2) of young flies treated with 10 mM t-BHP as in A. D) Relative activity (%) of 26S proteasome, showing proteasome activation in the gonads of young Drosophila exposed to 10 to 20 mM t-BHP as in B. E) Relative activity (%) of 26S proteasome in aged female fly somatic and ovarian tissues following exposure to 20 mM t-BHP for 3–4 d. F1, F2) Relative percentage of GFP fluorescence in young (Y) and old (O) female somatic and ovarian tissues of gstD-ARE:GFP and gstD-mARE:GFP transgenic flies exposed to 20 mM paraquat for 15 h (F1), and relative cncC and keap1 mRNA expression levels (F2) in somatic and ovarian tissues of 10- to 13-d-old female flies. GAPDH and rp49 were used as references for equal total protein and RNA input, respectively. Bars represent means ± sd; n = 3. *,#P < 0.05, **P < 0.01.

Figure 7.

CncC activation (by RNAi-mediated keap1 knockdown) in adult flies results in youthful proteasome gene expression patterns in the aged soma, whereas RNAi-mediated cncC knockdown induces a soma-like down-regulation of proteasome genes expression in ovaries of aged flies. A) Densitometric RT-PCR analyses of rpn11, α7, and β5 proteasome gene expression levels in somatic (left panel) or ovarian (right panel) tissues of young (Y) UAS keap1 (left panel) or UAS cncC (right panel) RNAi flies. RU486 was administered to flies for ∼7 d; control flies were treated with ethanol (EtOH). UAS, upstream activation sequence. B, C) RT-PCR densitometric analyses of the rpn11, α7, β5, and cncC gene expression levels (left panels) and representative agarose gels (right panels) in young (Y), middle-aged (M), and old (O) somatic tissues (B) or ovaries (C) of UAS keap1 (B) or UAS cncC (C) RNAi flies; in all cases, control flies were fed with EtOH. Eclosed flies were constantly fed with RU486 in order to induce transgene expression. As either keap1 or cncC RNAi affected maximum and median life span of flies (data to be reported elsewhere), in all cases, young flies used in these analyses were at the age of ≤15% of their life span, middle-aged at ∼45–55% of their life span, and old flies at ≥80% of their life span. Bars represent means ± sd; n = 2. Controls were arbitrarily set to 100%. *P < 0.05.