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. 2023 Apr 30;24(9):8100.
doi: 10.3390/ijms24098100.

The Small-Molecule Enhancers of Autophagy AUTEN-67 and -99 Delay Ageing in Drosophila Striated Muscle Cells

Marcell Komlós  1 , Janka Szinyákovics  1   2 , Gergő Falcsik  1 , Tímea Sigmond  1 , Bálint Jezsó  3   4 , Tibor Vellai  1   2 , Tibor Kovács  1
Affiliations

The Small-Molecule Enhancers of Autophagy AUTEN-67 and -99 Delay Ageing in Drosophila Striated Muscle Cells

Marcell Komlós et al. Int J Mol Sci. .

Abstract

Autophagy (cellular self-degradation) plays a major role in maintaining the functional integrity (homeostasis) of essentially all eukaryotic cells. During the process, superfluous and damaged cellular constituents are delivered into the lysosomal compartment for enzymatic degradation. In humans, age-related defects in autophagy have been linked to the incidence of various age-associated degenerative pathologies (e.g., cancer, neurodegenerative diseases, diabetes, tissue atrophy and fibrosis, and immune deficiency) and accelerated ageing. Muscle mass decreases at detectable levels already in middle-aged patients, and this change can increase up to 30-50% at age 80. AUTEN-67 and -99, two small-molecule enhancers of autophagy with cytoprotective and anti-ageing effects have been previously identified and initially characterized. These compounds can increase the life span in wild-type and neurodegenerative model strains of the fruit fly Drosophila melanogaster. Adult flies were treated with these AUTEN molecules via feeding. Fluorescence and electron microscopy and Western blotting were used to assess the level of autophagy and cellular senescence. Flying tests were used to measure the locomotor ability of the treated animals at different ages. In the current study, the effects of AUTEN-67 and -99 were observed on striated muscle cells using the Drosophila indirect flight muscle (IFM) as a model. The two molecules were capable of inducing autophagy in IFM cells, thereby lowering the accumulation of protein aggregates and damaged mitochondria, both characterizing muscle ageing. Furthermore, the two molecules significantly improved the flying ability of treated animals. AUTEN-67 and -99 decrease the rate at which striated muscle cells age. These results may have a significant medical relevance that could be further examined in mammalian models.

Keywords: AUTEN-67; AUTEN-99; Drosophila; EDTP; MTMR14; ageing; autophagy induction; muscle ageing.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Mechanisms by which AUTEN-67 and -99 activate autophagy. (A) AUTEN-67 and -99 can induce autophagic degradation through interfering with MTMR14/EDTP. Converting PI into PI3P is an essential step for the formation of the autophagic isolation membrane. This process is catalyzed by the Vps34 kinase complex. In mammals, MTMR14/Jumpy antagonizes with the complex. MTMR14/Jumpy is a shared target of the two AUTEN molecules used in this study. EDTP, the Drosophila orthologue of MTMR14/Jumpy, can also be blocked by these AUTEN molecules. (B) Striated muscle from a young and an old animal. In the old specimen, protein aggregates (yellow) and damaged mitochondria containing abnormal internal lamellas (blue) accumulate, and there are nuclei with a highly condensed chromatin structure (green).
Figure 2
Figure 2
AUTEN-67 and 99 are capable of inducing autophagy in striated muscle. (A,A″) The amount of Ref(2)P/p62 serving as a substrate of autophagy is inversely related to the capacity of the process. AUTEN molecules decrease the amount of Ref(2)P-positive aggregates in the striated muscle of 21-day-old animals maintained at 29 °C. Yellow arrowheads indicate Ref(2)P-positive structures characterizing normal tissues, whereas white arrowheads point to Ref(2)P-positive aggregates accumulating with age (A′,A″). The amount of mCherry-Atg8a-positive structures increases in animals treated with an AUTEN molecule, as compared with the untreated control. Nuclei are indicated by blue coloring (Hoechst staining). In the right panels, the area outlined with a dotted line is enlarged (A,A′). (B,B′) Western blot analysis showing that the amount of soluble Ref(2)P is also lowered in samples isolated from the IFM of animals at age of adult day 21. Difference between Atg8a-I (soluble) and Atg8a-II (membrane-bound) levels lowers in response to AUTEN treatments. αTub84B was used as an internal control. In panels (A′,B′), bars represent ±S.D. *: p < 0.05; **: p < 0.01; ***: p < 0.001. For statistics, see Section 3. (C) Transmission electron microscopic images showing enhanced levels of autophagy in the IFM of animals exposed to an AUTEN molecule. Arrows show autophagic structures, autophagosomes and autolysosomes. Animals were maintained at 29 °C.
Figure 3
Figure 3
AUTEN small-molecule enhancers of autophagy delay ageing in Drosophila striated muscle. (A) Transmission electron microscopic images showing aberrant inner membrane structures of mitochondria (red arrowheads) in striated muscle cells of aged animals. The amount of abnormal structures was lowered in samples treated with an AUTEN molecule. (B,B′) The overlap between mitochondria (mito-GFP—green) and autophagic structures (mCherry-Atg8a—red) increases in samples treated with AUTEN-67 or -99 as compared with the untreated control. In the right panel, the area outlined by a dotted line is enlarged (A). In panel (B′), bars represent ±S.D. *: p < 0.05; **: p < 0.01. For statistics, see Section 3. (C) Electron microscopic analysis showing large multilamellar bodies (MLB—shown by yellow arrowheads) in the IFM of 21-day-old animals. The presence of these structures is indicative of an abnormal lysosomal breakdown. Animals of the same age display lower amounts of MLBs in response to AUTEN treatments. Animals were maintained at 29 °C.
Figure 4
Figure 4
AUTEN-67 and -99 improve flying ability in Drosophila. (A) To perform a flying assay, a transparent, 80 cm high plexiglass measuring tube (a) was used. A paper covered by a transparent insect trap was placed on the inner wall of the tube. Glass vials containing animals were dropped in an inverted orientation into a drop tube on the top (b). The vial was stopped by a funnel (c) while animals kept falling into the measuring tube. Fitter animals flying towards the light got stuck to the paper covered by the insect trap close to the top of the tube, whereas old and unhealthy animals did the same close to the bottom of the tube. Animals unable to fly dropped into the paraffin oil (d) found at the bottom of the tube. Between the measurements, the insect trap was removed from the tube to determine the height at which they got stuck. Before the next measurement, animals were removed from the paper trap. The cleared trap was reinstalled inside the tube. (B) AUTEN treatments significantly improved the ability of both young (7 days) and aged (21 days) adults to fly. Animals were kept at 29 °C. (C) A hypomorphic allele of EDTP, EDTPMI08496, decreases the ability of 7 and 14 days old animals to fly. (D) EDTPMI08496 is a thermosensitive allele with a restrictive temperature of 29 °C. Development of animals occurred at a permissive temperature (18 °C) (in this way, potential developmental effects influencing flying ability were excluded); then, animals were transferred at 29 °C. Flying ability of mutant animals defective for EDTP function failed at the restrictive temperature (29 °C) only. Hence, defects in flying were not a consequence of lacking EDTP function during development. (E,E′) Proteins were extracted from animals treated as shown in panel (D). Western blot analysis reveals that amounts of EDTP lower only in EDTPMI08496 mutants maintained at 29 °C. The amount of Ref(2)P also decreased in these animals (maintained under a restrictive condition), but not in those maintained at 18 °C (a permissive condition). Similarly, the ratio of Atg8a-I/Atg8a-II levels in EDTPMI08496 mutants relative to control changed at 29 °C only. (F) Flying ability of control (treated with DMSO) and AUTEN-treated EDTPMI08496 animals at the adult age day 14. In the absence of EDTP function, AUTEN molecules cannot improve the ability of animals to fly. (G) Specificity of AUTEN-67 and -99 was also assessed by a Western blot analysis. Ref(2)P level decreased in EDTPMI08496 mutants, and this change cannot be influenced by an AUTEN treatment. Thus, these AUTEN molecules exert their effect on flying ability by inhibiting EDTP. In panels (B,D,E′,F), bars represent ±S.D. *: p < 0.05; **: p < 0.01; ***: p < 0.001. For statistics, see Section 3.
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