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

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.


Introduction
Autophagy is an evolutionarily conserved, lysosome-dependent, self-degradation process of eukaryotic cells by which superfluous and damaged cellular constituents can be effectively eliminated from the cytoplasm [1][2][3][4][5]. This catabolic process provides energy and monomers for synthetic processes [6]. Thus, autophagy is essential for maintaining cellular homeostasis. The capacity of the autophagic process exhibits a gradual decline during the adult life span, thereby contributing to the development of various age-related degenerative diseases (such as cancer, diabetes, muscle atrophy, tissue fibrosis, diverse forms of neurodegenerative processes and immune deficiency) [7][8][9]. The pharmacological 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).

AUTEN-67 and -99 Induce Autophagy in the Drosophila Striated Muscle
According to previous results, treatments of Drosophila with the AUTEN molecules (AUTEN-67 and -99) enhance the ability of animals to climb up on the wall of a glass vial [18,20]. In these earlier studies, however, enhanced autophagic activity upon AUTEN treatment was demonstrated only in the brain tissue of adult animals. It is reasonable to assume that improvement in moving ability can also result from enhanced autophagy in the muscle tissue. Hence, here, we studied the autophagic process in the IFM of control flies and animals treated by AUTEN-67 and -99 at different adult stages. AUTEN (which were dissolved in DMSO)-treated animals were compared with age-matched controls (exposed to DMSO only).
Mammalian p62/SQSTM1 (sequestrome 1), the single Drosophila orthologue of which is Ref(2)P (refractory to sigma P), serves as a substrate for autophagic degradation. Therefore, the level of p62/SQSTM1 inversely correlates with autophagic activity; the higher the intracellular level of the substrate, the lower the activity of the process [42]. To monitor autophagic activity, Ref(2)P levels were measured in the IFM at different adult stages, and we found that these levels increase as the animal ages (Figure 2A,A ). These results indicate that autophagic activity gradually lowers with age in the examined tissue. Similar data have been previously obtained from other Drosophila tissues, including the nervous system [30,43]. Next, Ref(2)P levels were compared in the IFM between control and AUTEN-67/99-treated animals. The results showed that fewer amounts of the substrate were detectable in the treated animals relative to the control at the same ages (Figure 2A), indicating that the molecules potently induce autophagy in these cells.
Damaged proteins are frequently marked with ubiquitin, and ubiquitinated protein aggregates are primarily degraded by autophagy (i.e., ubiquitinated proteins represent another substrate group for the autophagic breakdown) [42,44]. Thus, ubiquitin levels in the IFM during adulthood (at adult stages of 7 days-young and 21 days-aged) were also monitored. Ubiquitin levels also significantly increased in older animals relative to younger ones but decreased in flies treated with an AUTEN compound compared with untreated control at the same age ( Figure S1A,B ). Together, these data suggest that the capacity of autophagy in the IFM endogenously declines during ageing, and it can be enhanced by AUTEN molecules.
LC3B is another tractable marker for monitoring autophagic activity. The Drosophila orthologue of LC3B is Atg8a [16]. Atg8a binds to the PE of the phagophore, the initial structure of autophagosome formation [45]. Therefore, the protein can be found in the forming autophagic vesicle, and, later, in the inner membrane of the matured autophagosome. Atg8a is also degraded by autolysosomal enzymes [46]. An mCherry-Atg8a reporter readily labels early (phagophores and autophagosomes) and late (autolysosomes) autophagic structures [47]. This study found that AUTEN treatments increase the amount of mCherry-Atg8a-positive structures in the IFM. (Figure 2A ,A ). The number of lysosomal components can also be measured by labeling cathepsin-L hydrolase with a specific antibody [48]. The increased amounts of cathepsin-L-positive structures were detected in the IFM of animals treated with an AUTEN molecule as compared to untreated control at the same age ( Figure S1C,C ).  Fluorescence microscopy can directly reveal autophagic membranous structures, insoluble protein aggregates and abnormal organelles, while Western blotting is primarily suitable to detect the level of mainly soluble cytoplasmic proteins. Using the latter method, the amounts of soluble Ref(2)P and ubiquitinated proteins showed a significant decrease in 21-day-old animals treated with AUTEN molecules, as compared with the control at the same stage ( Figure 2B,B and Figure S2A,A ). The amounts of Atg8a isoform (I and II)positive structures were also examined. The soluble Atg8a-I is a 15 kDa large protein, while its lipidated form (termed PE-Atg8a or Atg8aII) is only 12 kDa in size [49]. Because Atg8a-II is also degraded by autophagy, the protein can also be considered an autophagic substrate. Upon autophagy activation, Atg8a-II levels increase. Thus, changes in autophagy activity can be inferred from the relative ratio of Atg8a-I and Atg8a-II proteins [49]. The current study found that the application of AUTEN-67 and AUTEN-99 each can decrease the ratio of Atg8a-I/Atg8a-II proteins compared to age-matched control ( Figures 2B and S2B).
The effect of these small molecules on autophagic activity was also studied at the ultrastructural level, and increased numbers of autophagosomes and autolysosomes were observed in the IFM samples exposed to AUTEN treatment ( Figure 2C). Thus, AUTEN molecules are able to induce autophagy in the Drosophila IFM.

AUTEN Molecules Decrease the Amount of Damaged Mitochondria in the IFM
Ageing in striated muscle can also be characterized by the accumulation of damaged, non-functional mitochondria, which are often wedged into protein aggregates [50,51]. Using transmission electron microscopy, the IFM was examined in young (7 days old) and old (21 days old) animals treated with an AUTEN molecule. This study found that samples from old flies contain higher amounts of damaged mitochondria than those derived from younger animals ( Figure 3A). Furthermore, AUTEN treatment significantly decreased the number of mitochondria with abnormal inner lamellas as compared to untreated age-matched controls ( Figure 3A). The elimination of damaged mitochondria by selective autophagy is called mitophagy [52]. For studying the mitophagic process, a colocalization assay was performed of mCherry-Atg8a-and mito-GFP-positive structures. The results showed that colocalization of these markers is enhanced in AUTEN-treated IFM samples relative to untreated age-matched controls ( Figure 3B,B ). In samples from aged (21 days old) animals, multilamellar bodies (MLB) were observed, indicating a partial digestion of mitochondria and late endosomes containing intact inner membranes ( Figure 3C) [53]. In the untreated IFM samples, significantly larger MBLs were detected as compared to treated ones ( Figure 3C). The presence of enlarged MBL structures may refer to compromised lysosomal degradation [53]. It can be concluded that AUTEN molecules confer a beneficial effect on the elimination of damaged mitochondria. mitochondria and late endosomes containing intact inner membranes ( Figure 3C) [53]. In the untreated IFM samples, significantly larger MBLs were detected as compared to treated ones ( Figure 3C). The presence of enlarged MBL structures may refer to compromised lysosomal degradation [53]. It can be concluded that AUTEN molecules confer a beneficial effect on the elimination of damaged mitochondria.

AUTEN Molecules Improve Flying Ability in Drosophila
Next, the functional activity of striated muscle was tested in AUTEN-treated animals. The effect of AUTEN molecules on movement was assessed by using a flight test. In this method, selected animals are dropped into a long transparent tube, the wall of which is covered with an insect trap. In the tube, animals switch from falling to flying (flying toward the light) and get stuck in the trap. Fitter animals stick out at the top of the tube, while aged or abnormally flying flies stick out closer to the bottom of the tube. Less-flying animals fall into a paraffin oil collection container under the pipe ( Figure 4A) [54]. The current study showed that AUTEN molecules increase the flying ability of both young and aged flies ( Figure 4B). The effects of the thermosensitive hypomorphic mutant EDTP MI0849 (hereafter referred to as EDTP MI ) were also tested. EDTP MI mutant adults died significantly earlier at 29 • C than the control ( Figure 4C), so the analysis could only be performed on animals aged 7 and 14 days old. As expected, EDTP MI mutants flew less than agematched controls ( Figure 4C). Thus, pharmacological and genetic inhibitions of EDTP led to opposing results. This implies that a developmental role of EDTP influences flying ability in adults. AUTEN treatments were performed only at adult stages, while the mutation affects EDTP function throughout the entire lifespan, including development. Therefore, flying assays on EDTP MI mutants were repeated in a specific way; animals were maintained under permissive conditions (at 18 • C) during development, then transferred to 29 • C (restrictive condition) throughout adulthood. A decline in flying ability was also detectable in mutant animals ( Figure 4D). Under these conditions, further Western blot analysis confirmed that the amount of EDTP proteins in the EDTP MI genetic background does not decrease at 18 • C; in line with this finding, the amounts of the autophagic substrates Ref(2)P and Atg8a-II remained unchanged compared to the control kept under the same conditions ( Figures 4E,E and S3A). Under restrictive conditions, however, the amounts of EDTP and Ref (2) decreased significantly ( Figures 4E,E and S3A). Thus, decreased moving ability caused by EDTP deficiency is partially independent of the developmental role of EDTP. Finally, the specificity of AUTEN-67 or -99 to EDTP was tested in the muscle. After treatment with AUTEN-67 and -99, the flying abilities of EDTP MI mutants were measured. The flying ability of treated mutants was not changed as compared with untreated controls ( Figure 4F). In this study, the autophagic activity was also observed in EDTP MI mutants treated with an AUTEN. Another Western blot analysis confirmed that EDTP protein levels were lowered in these mutants relative to the control genetic background ( Figure 4G). Consistent with these data, the amount of Ref(2)P was also lower in the control and treated mutants ( Figure 4G). So, treatment with an AUTEN cannot further increase autophagic activity in EDTP MI mutant animals. These results suggest that AUTEN-67 and -99 are specific to EDTP. ol. Sci. 2023, 24, x FOR PEER REVIEW 9 of 16

Discussion
AUTEN-67 and AUTEN-99 appear to act as specific inhibitors of MTMR proteins, and previous research uncovered AUTEN-type small-molecule enhancers of autophagy as potent agents in treating various neurodegenerative pathologies [18][19][20]. Mutational inactivation of MTMRs leads to muscle-related diseases in humans, raising the possibility that AUTENs may also contribute side effects in the muscle tissue [35,54]. In the present study, the effects of AUTEN-67 and -99 were examined on the age-related functionality of Drosophila striated muscle cells and excluded this negative effect.
Using fluorescent and electron microscopy, as well as Western blot analyses, this study showed that autophagy is activated in the IFM of animals treated with an AUTEN molecule ( Figure 2 and Figure S1). AUTEN-67 and -99 had similar effects on autophagy activation. During ageing, unfunctional mitochondria accumulate in the muscle tissue and may significantly interfere with its working capacity [55]. Moreover, damaged mitochondria can produce reactive oxygen species serving as cellular toxins [56]. The accumulation of tangled mitochondria can also lead to mitochondrial myopathies, which can lead to weakness, muscular dysfunction and a host of other symptoms [57]. Vesicle nucleation promoted by AUTEN molecules was likely to contribute to the degradation of damaged mitochondria. This is supported by the result that upon treatment with an AUTEN molecule, autophagy structures significantly overlapped with labeled mitochondria ( Figure 3B,B ), and that in the IFM of aged animals, there were fewer damaged mitochondria containing abnormal lamellas than in controls ( Figure 3).
The current study also measured the effect of AUTEN molecules on muscle function by using a flying assay. Consistent with previously published data from climbing assays [18,20], AUTEN treatments improved the ability of Drosophila to fly ( Figure 4B). In contrast to this pharmacological inhibition, flying ability was found to be significantly worse in EDTP hypomorphic mutant animals compared to wild-type ones ( Figure 4C). It is worth noting that this change was not influenced by development because EDTP functions were intact until the onset of adulthood (hypomorphic EDTP MI08496 mutants were maintained at a permissive temperature during development) (Figure 4C,D). Thus, it can be concluded that pharmacological (AUTEN-mediated) inhibition of EDTP reduces, but does not eliminate, the function of the protein. This can moderately induce autophagic activity; that is, AUTEN treatment can restore the process to basal (physiological) levels.
Differences in results between pharmacological and genetic inhibitions of EDTP show that these AUTEN molecules are not exclusively specific to EDTP; rather, they may also interfere with other myotubularin-related lipid phosphatases [29,35]. This prompted us to monitor the specificity of AUTENs in the IFM. Both AUTEN-67 and -99 failed to improve flying ability in EDTP-defective mutant animals and consistently did not increase autophagic and flying activities in the IFM of EDTP MI08496 mutant animals ( Figure 4F,G). Thus, these small-molecule enhancers of autophagy appear to specifically affect EDTP in the IFM of adult animals.
Although the Drosophila IFM is a tractable model for studying muscle ageing, the system possesses several limitations [58]. For example, muscle regeneration characterizing mammalian organisms, as well as the age-related loss of muscle mass, cannot be examined in the fruit fly [59][60][61][62]. As in the nervous system, the IFM has been shown to increase EDTP expression with age [30,31,63]. If the age-dependent increase in EDTP expression causes harmful protein dephosphorylation in muscle, it may be important to investigate the effect of AUTENs on phosphatase activity in IFM ageing in a future study. Hence, further studies should analyze whether AUTENs can also delay muscle ageing and influence the above processes in mammalian systems. If these compounds positively affect muscle functions at advanced ages, they will serve as potent drug candidates for inhibiting the progression of diverse age-associated symptoms.
We previously demonstrated that in Drosophila, AUTEN-67 and -99 are capable of inducing the autophagic process in neural tissue [18,20], and can also enhance the capacity of the process in larval fat body cells [19,20]. In this study, evidence was provided that these small molecules are able to induce autophagy in IFM cells, thereby delaying ageing in this tissue. In the future, it is worth assaying the autophagy-inducing effect of AUTEN molecules in other tissues and disease models.

Drosophila Strains
Drosophila strain stocks were maintained on standard cornmeal-sugar-agar medium at 25 • C, and experiments were carried out at 28 • C unless noted. Strains were obtained from the Bloomington Drosophila Stock Center (BDSC) or kindly provided by other researchers.
The following alleles were used (strains):

Flying Assays
Animals were anesthetized with CO 2 , and rested for 1 h before the flying assay. Vials containing the flies were dropped into a 25 cm long drop tube, the bottom of which was narrowed with a funnel. Animals continued to fall through this opening. The measuring tube was 80 cm long; a paper covered by a transparent insect trap was placed on the inner wall of this tube ( Figure 3A). Falling animals try to switch to flight and flee towards the light, which is the side of the tube because of the transparent paper. Fitter animals stick closer to the top of the tube, while old animals stick closer to the bottom of the tube or fall into a container that contains paraffin oil below the tube. To determine flying ability, the height at which they got stuck (distance from the bottom of the funnel) was recorded for each animal. Trapped animals were removed from the paper and oil before the next measurement [68].

Evaluation and Statistics
For the statistical analysis of flying measurement, Western blot and fluorescence microscopy, R Studio (Version 3.4.3), were used. The distribution of samples (normal or not) was tested with a Lilliefors test. If it was normal, the F-test was performed to compare variances. In case variances were equal, a two-sample t-test was used; otherwise, a t-test for unequal variances was applied. In the case of non-normal distribution, a Mann-Whitney U-test was performed. On the plot, the boxes represent the most typical 50% of the samples, the lines indicate the median and the upper and lower whiskers show the remaining 25-25% of the samples. Circles mark outliers. p < 0.05 *; p < 0.01 **; p < 0.001 ***.

Conclusions
These results show that both AUTEN-67 and AUTEN-99 can increase autophagy in the striated muscles of animals and thus contribute to the improvement of movement function. The drug treatments were successful in reducing morphological changes in the aged mus-