Functional wiring of proteostatic and mitostatic modules ensures transient organismal survival during imbalanced mitochondrial dynamics

Abstract

Being an assembly of protein machines, cells depend on adequate supply of energetic molecules for retaining their homeodynamics. Consequently, mitochondria functionality is ensured by quality control systems and mitochondrial dynamics (fusion/fission). Similarly, proteome stability is maintained by the machineries of the proteostasis network. We report here that reduced mitochondrial fusion rates in Drosophila caused developmental lethality or if induced in the adult accelerated aging. Imbalanced mitochondrial dynamics were tolerable for various periods in young flies, where they caused oxidative stress and proteome instability that mobilized Nrf2 and foxo to upregulate cytoprotective antioxidant/proteostatic modules. Consistently, proteasome inhibition or Nrf2, foxo knock down in young flies exaggerated perturbed mitochondrial dynamics toxicity. Neither Nrf2 overexpression (with concomitant proteasome activation) nor Atg8a upregulation suppressed the deregulated mitochondrial dynamics toxicity, which was mildly mitigated by antioxidants. Thus, despite extensive functional wiring of mitostatic and antioxidant/proteostatic modules, sustained loss-of mitostasis exhausts adaptation responses triggering premature aging.

Keywords: Aging; Drp1; Mitofusins; Mitostasis; Opa1; Proteostasis.

Graphical abstract

Fig. 1

Opa1 KD disrupts mitochondria structure and function; induces metabolic and neuromuscular defects and drastically accelerates aging. (A, B) CLSM visualization of Mito^GFP in larval muscle (A; Gal4^Mef2) or nervous system (B; Gal4^D42) after Opa1 OE or KD. (C) Relative mitochondrial ST3/ST4 respiratory efficiency rates from somatic tissues of flies expressing (or not) the shown transgenes; young mated flies were exposed to RU486 for 7 days. (D) Oxygen consumption values (basal and after Oligomycin addition) during mitochondrial respiration assays. (E) Relative (%) content of GLU, TREH and GLY in larvae tissue after muscle specific (Gal4^Mef2) Opa1 OE or KD. (F) CLSM visualization of larvae fat body after BODIPY staining of lipids in shown Opa1 transgenic lines (Gal4^Mef2). (G) Climbing activity (%) of middle aged transgenic flies of the indicated genotypes. (H, I) Longevity curves of the indicated transgenic lines; shaded rectangles and shown periods in days indicate the duration of neutral (vs. control) effect of the expressed transgene; viability at 20 days post-Opa1 KD was ∼28%. (J) Longevity curves following induced ubiquitous Opa1 KD in middle aged (30 days old) flies; viability at 20 days post-transgene expression was ∼20%. Statistics of the longevity curves are reported in Table S1. In (C, D), (G–J) the Gal4^GS−Tub inducible ubiquitous driver was used. Bars, ± SD; n ≥ 2; *P < 0.05; **P < 0.01.

Fig. 2

KD of Opa1 is increasingly toxic in male flies. (A) CLSM visualization of mitochondria (Mito^GFP) in larvae muscles (Gal4^Mef2) of male and female flies. (B) Relative mitochondrial ST3/ST4 and FCCP/ST4 values after Opa1 KD in adult male and female flies. (C) EM visualization of mitochondria and myofibril structure after Opa1 KD; arrows indicate disrupted cristae and the rectangle aggregated mitochondria. In (B), (C) the transgene was induced in young mated flies for 7 days. (D) Longevity curves of male and female flies after inducible ubiquitous Opa1 KD. Statistics of the shown longevity curves are reported in Table S1. In (B–D) the Gal4^GS−Tub driver was used. Bars, ± SD; n ≥ 2; *P < 0.05; **P < 0.01.

Fig. 3

Imbalanced mitochondrial dynamics towards reduced fusion causes oxidative stress and proteome instability triggering the activation of antioxidant/proteostatic modules. (A) Relative (%) ROS levels in tissues of young flies after ubiquitous inducible expression of the shown transgenes. (B) CLSM visualization of the Mito^Timer reporter in the nervous system of control (+/Gal4^Elav) larvae or following Marf or Opa1 KD. (C) Representative immunoblot analysis of tissue protein samples probed for total protein ubiquitination (Ub) and carbonylation (DNP). (D) Relative (%) activation of AREs in larvae following muscle targeted KD of Opa1 (Gal4^Mef2). (E) Representative immunoblot analysis of young flies' tissue protein samples probed with antibodies against proteasomal subunits 20S-α and Prosβ5 . (F, G) Relative (%) proteasomal (F) and cathepsins B, L (G) activities in young flies' tissues expressing (or not) ubiquitously the shown transgenes. In (C), (E) GAPDH was used as reference. In (A), (C), (E-G) the transgene was induced in young flies for 7 days by using the Gal4^GS−Tub driver. Bars, ± SD; n ≥ 2; *P < 0.05; **P < 0.01.

Fig. 4

cncC/Nrf2 and foxo mediate cytoprotective cellular responses after Marf KD in young flies; their inactivation enhances Marf KD-mediated lethality. (A) Relative expression of shown genes in young flies after combined ubiquitous expression (or not) of the indicated transgenes. (B–D) Relative (%) ROS levels (B), proteasome activities (C), proteome ubiquitination (GAPDH was used as loading reference) (D) and cathepsins B, L (E) activity in shown transgenic lines. (F) CLSM viewing of mitochondria (Mito^GFP) in larvae muscle (Gal4^Mef2) of the indicated genotypes. (G) Relative quantification of mitochondria density (number/area) and length in larvae muscles of the indicated genotypes. (H, I) Longevity curves of the indicated transgenic lines; shaded rectangles highlight the accelerated toxicity (vs. Marf KD flies) in young flies co-expressing the indicated transgenes. Statistics of the shown longevity curves are reported in Table S1. In (A–E) the transgene was induced in young flies for 7 days. In (A–E), (H), (I) the Gal4^GS−Tub driver was used. Gene expression was plotted vs. the respective control set to 1; the RpL32/rp49 gene expression was used as input reference. Bars, ± SD; n ≥ 2; *P < 0.05; **P < 0.01.

Fig. 5

Cytoprotective cell responses after Opa1 KD in young flies are mediated by cncC/Nrf2 and foxo; combined Opa1 and cncC/Nr2 or foxo KD exaggerates the Opa1 KD-mediated toxicity. (A) Relative expression of shown genes in young flies following combined ubiquitous expression (or not) of the shown transgenes. (B–E) Relative (%) ROS levels (B), proteasome activities (C), proteome ubiquitination (GAPDH was used as loading reference) (D) and cathepsins B, L (E) activity in the shown transgenic lines. (F) CLSM viewing of Mito^GFP in larvae muscle (Gal4^Mef2) of the indicated genotypes. (G) Relative quantification of mitochondria density (number/area) and length in larvae muscles of the indicated genotypes. (H, I) Longevity curves of the shown transgenic lines; shaded rectangles highlight the accelerated toxicity (vs. Opa1 KD) in young flies co-expressing the shown transgenes. Statistics of the longevity curves are reported in Table S1. In (A–E) transgenes were induced for 7 days. In (A–E), (H), (I) the Gal4^GS−Tub driver was used. Gene expression was plotted vs. the respective control set to 1; the RpL32/rp49 gene expression was used as input reference. Bars, ± SD; n ≥ 2; *P < 0.05; **P < 0.01.

Fig. 6

Combined cncC/Nrf2 OE and Marf or Opa1 KD activates cytoprotective antioxidant/proteostatic modules; yet, it cannot mitigate the imbalanced mitochondrial dynamics-mediated toxicity. (A–C) Relative (%) ROS levels (A), proteasome (B) and cathepsins B, L (C) activities of the shown young transgenic flies. (D) Immunoblot analysis of young flies' tissue protein samples probed with antibodies against proteasomal subunits 20S-α and Prosβ5; GAPDH was used as reference. (E) CLSM imaging of mitochondria (Mito^GFP) in larvae muscle (Gal4^Mef2) of the indicated genotypes. (F) Relative quantification of mitochondria density (number/area) and length in larvae muscle (Gal4^Mef2) of the indicated genotypes. (G, H) Longevity curves of the indicated transgenic lines; the shaded rectangle in (G) highlights the increased toxicity seen in young flies after cncC/Nrf2 OE at the Marf KD background. In (A–D) flies were treated with RU486 for 7 days. Statistics of the longevity curves are reported in Table S1. In (A–D), (G), (H) the Gal4^GS−Tub driver was used. Bars, ± SD; n ≥ 2; *P < 0.05; **P < 0.01.

Fig. 7

Treatment with 6BIO and Tiron reduce oxidative load and activate antioxidant responses at the Marf and Opa1 KD genetic backgrounds. AREs-GFP (A), ROS levels (B) and proteasome activities (C) following ubiquitous Marf or Opa1 KD and treatment (or not) with 6BIO or Tiron. (D, E) CLSM visualization of the cyto-roGFP-Orp1 (D) and mito-roGFP2-Orp1 (E) reporters in larvae muscle, after ubiquitous expression (Gal4^Tub) of Marf or Opa1 RNAi transgenes and co-treatment with Tiron. (F, G) Relative expression levels of proteostatic and mitostatic genes in somatic tissues of transgenic flies after ubiquitous expression of Marf (F) or Opa1 (G) RNAi transgenes and co-treatment with 6BIO or Tiron. (H–M) Longevity curves of the indicated transgenic flies after treatment with Tiron (H, I), 6BIO (J, K), or Catalase co-overexpression (L, M); statistics of the shown longevity curves are reported in Table S1. 6BIO was used at a concentration of 200 μM and Tiron of 5 mM. In (A–C), (F–M) the Gal4^GS−Tub driver was used. Bars, ± SD; n ≥ 2; *P < 0.05; **P < 0.01.