Alterations in Organismal Physiology, Impaired Stress Resistance, and Accelerated Aging in Drosophila Flies Adapted to Multigenerational Proteome Instability

Abstract

Being an assembly of highly sophisticated protein machines, cells depend heavily on proteostatic modules functionality and on adequate supply of energetic molecules for maintaining proteome stability. Yet, our understanding of the adaptations induced by multigenerational proteotoxic stress is limited. We report here that multigenerational (>80 generations) proteotoxic stress in Oregon^R flies induced by constant exposure to developmentally nonlethal doses of the proteasome inhibitor bortezomib (BTZ) (G80-BTZ flies) increased proteome instability and redox imbalance, reduced fecundity and body size, and caused neuromuscular defects; it also accelerated aging. G80-BTZ flies were mildly resistant to increased doses of BTZ and showed no age-related loss of proteasome activity; these adaptations correlated with sustained upregulation of proteostatic modules, which however occurred at the cost of minimal responses to increased BTZ doses and increased susceptibility to various types of additional proteotoxic stress, namely, autophagy inhibition or thermal stress. Multigenerational proteome instability and redox imbalance also caused metabolic reprogramming being evidenced by altered mitochondrial biogenesis and suppressed insulin/IGF-like signaling (IIS) in G80-BTZ flies. The toxic effects of multigenerational proteome instability could be partially mitigated by a low-protein diet that extended G80-BTZ flies' longevity. Overall, persistent proteotoxic stress triggers a highly conserved adaptive metabolic response mediated by the IIS pathway, which reallocates resources from growth and longevity to somatic preservation and stress tolerance. Yet, these trade-off adaptations occur at the cost of accelerated aging and/or reduced tolerance to additional stress, illustrating the limited buffering capacity of survival pathways.

Figure 1

Multigenerational developmentally nonlethal proteasome inhibition in Drosophila flies reduced fecundity and body size, caused neuromuscular defects, and accelerated aging. (a) Laid embryos (%) during a period of 24 h by young NT, NT-BTZ, G80, and G80-BTZ females. (b) Hatched flies (%) 14 days posttransferring thirty embryos per population to the respective culture medium. (c1) Images of female and male flies of the NT and G80-BTZ groups. (c2) Area (%) of right and left wings dissected from young female or male flies of the NT and G80-BTZ populations. (c3) Body weight (%) of middle-aged female or male flies collected from the NT and the G80-BTZ groups. (d) Locomotion (climbing) activity of young NT and G80-BTZ flies. (e) Longevity curves of female and male NT and G80-BTZ flies (e1) or of NT and G80 flies (e2); in (e2), equal numbers of female/male flies were used. Comparative statistics of the longevity assays are reported in Table S1. Bars, ±SD (n ≥ 2). ^∗ P < 0.05; ^∗∗ P < 0.01.

Figure 2

G80-BTZ flies were mildly resistant to BTZ. (a) Relative (%) CT-L and C-L proteasome activities in somatic tissues of NT-BTZ and G80-BTZ flies (see Fig. S1) following exposure to 1 μΜ ΒΤZ for 4 days (4D), 10 days (10D), or 20 days (20D). (b) Representative immunoblotting analysis of ubiquitin (Ub) levels in somatic tissue lysates of shown young flies' groups (compared to control samples from NT flies shown in Fig. S2B1); GAPDH probing was used as input reference. (c) Stereoscopic images of 3rd instar larvae and late-stage pupae of indicated groups. (d) Longevity curves of female and male NT-BTZ and G80-BTZ flies exposed to 1 μΜ ΒΤZ. Bars, ±SD (n ≥ 2). ^∗ P < 0.05.

Figure 3

Multigenerational proteotoxic stress triggered the upregulation of proteostatic modules. (a) Relative expression of Rpn11, Rpn10, Rpn6, Prosα7, Prosβ5, Prosβ2, and Prosβ1 proteasomal genes in somatic tissues of young NT, G80, and G80-BTZ flies; gene expression was plotted vs. the respective control (NT flies). (b) Representative immunoblotting analysis of 20S-α and Prosβ5 proteasomal subunit expression levels in somatic tissues of young female and male NT and G80 flies cultured (or not) in medium containing the indicated BTZ concentrations. (c) Relative (%) CT-L and C-L proteasome activities in young (Y) or aged (old; O) (≥80% of their lifespan) female/male flies of the shown groups. (d1) Relative expression of ref(2)P, Atg6, and Atg8a genes in somatic tissues of young NT and G80-BTZ flies; gene expression was plotted vs. the respective control NT flies. (d2) Relative (%) cathepsin B, L activity in somatic tissues of young NT, G80, and G80-BTZ flies. (e) Longevity curves of female (e1) and male (e2) flies of the indicated groups exposed to 200 μΜ of the autophagy inhibitor chloroquine (CQ). The Rp49 gene expression was used in (a), (b) as input reference. Bars, ±SD (n ≥ 2); ^∗ P < 0.05; ^∗∗ P < 0.01.

Figure 4

Sustained proteome instability caused metabolic reprogramming in G80-BTZ flies being evidenced by altered mitochondrial biogenesis. (a) CLSM visualization following immunofluorescence staining of young flies' thoracic muscle tissues with ATP5a antibody; samples were counterstained with DAPI. (b) Relative expression levels of mitochondrial biogenesis (PGC-1, TFAM) and chaperones (Hsp10, Hp60A, and Hsc70-5) genes in shown young flies' somatic tissues. (c1) Relative expression levels of mitochondrial energetics (SdhA, ATPsynβ), quality control (Lon), dynamics (Marf, Opa1, and Drp1), and mitophagy (park, Pink1) genes in indicated young flies' somatic tissues. (c2) ATP5a expression levels (blue native-PAGE; upper panel) and relative (%) quantitation (lower panel) in isolated mitochondria from somatic tissues of young female and male flies; GAPDH probing in cytosolic preparations was used as an input reference. (d) Mitochondrial ST3/ST4 respiratory ratio in somatic tissues of the indicated flies' groups. Gene expression was plotted vs. the respective control, and Rp49 gene expression was used as input reference. Bars, ±SD (n ≥ 2). ^∗ P < 0.05; ^∗∗ P < 0.01.

Figure 5

Multigenerational proteome instability in G80-BTZ flies induced metabolic reprogramming being evidenced by suppressed insulin/IGF-like signaling (IIS). (a) Relative (%) content of glucose (GLU), glycogen (GLY), and trehalose (TREH) levels in somatic tissues of female/male flies (10 or 20 days old) of the NT and G80-BTZ groups. (b1) CSLM visualization of fat bodies' (microdissected from 20-22-day-old female flies of the NT and G80-BTZ groups) lipid content after BODIPY staining; samples were also stained with a GLY antibody and counterstained with DAPI. (b2) Relative (%) size of lipid droplets shown in (b1). (c) Relative expression of Ilp2, Ilp6, InR, Pdk1, Akt1, foxo, G6P, Pepck, GlyP, GlyS, Ide, PyK, PEK, Akh, ATGL, and tgl genes in somatic tissues of flies of the NT and G80-BTZ populations. Gene expression was plotted vs. the respective control; the Rp49 gene expression was used as input reference. (d) Immunoblotting analysis of protein expression in female somatic tissues (d1), haemolymph (d2), or dissected heads (d3) of NT and G80-BTZ flies; blots were probed with antibodies against p-GSK3^S21/S9, sgg/GSK3, and foxo (d1); Ilp2 (d2); and Ilp2 and ImpL2 (d3). GAPDH or Ponceau S staining was used as loading reference. (e) Schematic representation of the IIS regulatory pathway in the context of Nrf2 and Foxo regulation. Bars, ±SD (n ≥ 2). ^∗ P < 0.05; ^∗∗ P < 0.01.

Figure 6

The toxicity of multigenerational proteotoxic stress can be partially mitigated by a low-protein-content diet; G80-BTZ flies were sensitive to thermal stress. (a1) Longevity curves of G80-BTZ flies fed (or not) with low-protein-content medium (LPM). (a2) Relative expression of the ref(2)P and Atg8a genes in NT or G80-BTZ flies fed (or not) with LPM. (b) Relative gene expression of proteasomal (Prosβ5, Keap1), antioxidant (Trxr-1), and autophagy-related (ref(2)P) cncC/Nrf2 transcriptional targets in somatic tissues of young G80-BTZ flies cultured for 5 days in medium containing (or not) 400 μΜ 6BIO. (c) Recorded (%) female or male paralyzed flies (c1) following exposure for 10 min to 40°C and rate (%) of recovery (c2) at room temperature. (d) Rate of flies' recovery (%) after vortexing (bang assay) for 20 seconds. Gene expression was plotted vs. the respective control and rp49 gene expression was used as input reference. Bars, ±SD (n ≥ 2). ^∗ P < 0.05; ^∗∗ P < 0.01.