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

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 OregonR 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.

genes in somatic tissues of aged NT and G80-BTZ flies; gene expression was plotted vs. the respective control (NT flies). (B 1 ) Staining with LysoTracker TM of isolated fat bodies from NT or G80-BTZ larvae and (B 2 ) relative (%) lysosome quantification per µm 2 , (n) nucleus. The Rp49 gene expression was used in (A) as input reference. Bars, ± SD (n=2); * P < 0.05. Figure S5. Proteome carbonylation and ubiquitination in mitochondria isolated from NT and G80-BTZ adult flies. Blots were probed with antibodies against DNP (carbonylation) and Ub; ATP5a and GAPDH were used a loading references in mitochondria and cytosol samples respectively.  Table S1; the shown G80-BTZ longevity curve is identical in (A), (B).  Table S1.
Graphical Abstract. Our summarized findings indicate that multigenerational proteotoxic stress and redox imbalance causes metabolic reprogramming and impaired stress resistance; it also promotes fecundity and neuromuscular defects, and accelerates aging. Tables   Table S1. Summary of lifespan experiments.

Tissues dissection, sorting of flies and haemolymph extraction
Proteasome is regulated in a tissue, sex and age-dependent manner [26]; thus, experiments were performed in dissected female or male somatic (head and thorax) tissues collected from young, middle-aged or old flies. For sorting experiments, female and male flies were anesthetized using CO 2 24 h prior to the experiment and the same number of individuals was used per sample. Haemolymph was isolated as described previously (Tsakiri et al., 2013).

Total RNA extraction and quantitative Real-Time PCR (Q-RT-PCR) analyses
Total RNA was extracted from flies' somatic tissues and converted to cDNA with the OneScript® cDNA Synthesis Kit of ABMGOOD (G234). cDNA was then subjected to Q-RT-PCR analysis using the HOT FIREPol® EvaGreen® qPCR Mix Plus of SOLISBIODYNE (08-24-00001) as described previously [21,33]. Primer sets were as described before [8,33,46].

Extraction of protein; immunoblot analyses and detection of protein carbonyl groups
Pooled or sex-sorted flies' somatic tissues or isolated mitochondria were homogenized on ice in min at 37°C. Next, gels were exposed to UV and fluorescent bands were visualized by standard gelimaging systems. For immunoblotting, remaining proteins were transferred to polyvinylidene fluoride membranes and immunoblotting was performed as described above. ROS levels, cathepsin and proteasome activities were measured as described previously [26,30, Tsakiri et al., 2013) and expressed as (%) values vs. respective controls. When both male/female samples were analyzed equal numbers of male/female flies were used.

Mitochondria isolation, measurement of mitochondrial respiration, blue native-PAGE and measurement of GLU, TREH and GLY Levels
Mitochondria isolation, respiration analyses and blue native PAGE were performed as described before (Nijtmans et al., 2002;Ferguson et al., 2005;Cogliati et al., 2013); in most cases dissected somatic tissues from 30 flies were analyzed. GLU, GLY and TREH levels from indicated tissues were measured as described previously [46] (Barrio et al., 2014). At least 3 replicates per genotype or experimental condition were performed.

Adipose and muscle tissue preparation for immunohistochemistry and CLSM viewing
Adipose tissue was attached to the dorsal abdominal area and was isolated after removing head, Nomenclature used for nontreated Oregon R flies (NT) cultured in standard medium (SM) and transiently exposed to 0.5 or 1 μM BTZ Nomenclature used for Oregon R flies cultured in SM containing 0.5 μM BTZ for >80 generations (G80-BTZ) and transiently exposed to BTZ-free SM or to SM containing 1 μM BTZ