Gut-brain axis neurodegeneration in a Drosophila model of Parkinson’s disease is linked to mitochondrial dysfunction

The innate immune response mounts a defence against foreign invaders, but the inappropriate induction of an innate immune response can cause diseases. Previous studies have provided ample evidence showing that mitochondria can be repurposed to promote inammatory signalling. Damaged mitochondria can also trigger inammation and promote diseases. Mutations in pink1 cause early-onset Parkinson’s disease (PD), and studies using Drosophila melanogaster have shown that pink1 mutants accumulate damaged mitochondria. Here, we showed that defective mitochondria in pink1 mutants activate Relish targets and demonstrated that inammatory signalling causes intestinal dysfunction in pink1-mutant ies. These effects result in the death of intestinal cells and metabolic reprogramming, which leads to neurotoxicity. We found that Relish signalling is activated downstream of a pathway stimulated by cytosolic DNA. The suppression of Relish in the intestinal midgut of pink1-mutant ies restores mitochondrial function and protects neurons in the brain. We thus conclude that the gut-brain axis causes neurotoxicity in a y model of PD through a mechanism involving mitochondrial dysfunction.


Introduction
Animals use the innate immune system as a defence against foreign invaders, which allows a rapid reaction to invading pathogens, such as microbes. Microbes often release foreign nucleic acids into the cytosol of infected cells, which can trigger innate immunity.
Mitochondria are energy-generating organelles that evolved from endosymbionts related to bacteria.
Mitochondria retain some of their genetic material in the form of DNA (mtDNA). mtDNA can trigger in ammatory responses similarly to bacterial DNA, and these responses are associated with the absence of methylated CpG sequences in both bacterial DNA and mtDNA 1 .
Cells safeguard the health of their mitochondria by operating several quality control (QC) mechanisms that ensure the disposal of faulty mitochondria. The organellar QC of organelles involves the degradation of defective mitochondria via mitophagy, which is a form of autophagy (reviewed in 2 ). Mutations in the gene encoding the mitochondrial kinase PINK1 lead to the accumulation of defective mitochondria and cause a form of familial Parkinson's disease (PD) (reviewed in 2 ). PINK1 functions in a molecular pathway that ensures the degradation of faulty mitochondria via mitophagy. In mice, the blockage of mitophagy induces the escape of mtDNA into the cytosol, where it activates innate immunity 3 .
In the fruit y (Drosophila melanogaster), the innate immune system consists of two branches (reviewed in 4 ): the Toll signalling pathway and the immune de ciency (Imd) pathway. The Imd pathway activates the Rel/NF-kB transcription factor Relish, which controls the expression of several antimicrobial peptides and is indispensable for normal immunity in ies. Relish rewires the metabolism by attenuating FOXOmediated lipolysis 5 and can also activate programmed cell death 6 .
The main immune organs in Drosophila are the fat body, which is considered to be equivalent to both vertebrate adipocytes and the liver, and the intestine. The intestine, in particular, plays a key role in activating the Imd pathway (reviewed in 7 ).
Mounting an immune response is energetically costly and requires trade-offs with other important biological functions (reviewed in 8 ), and in ies, this trade-off is important during starvation. In such settings, relish mutants exhibit increased survival when deprived of food 9 . The activation of Relish in response to cytosolic DNA can be mediated by interactions with Drosophila Eya, which is a molecule that maintains Relish in the cytoplasm 10 .
Here, we found that in pink1-mutant ies, in which mitophagy is blocked, Relish was activated with neurotoxic consequences. The accumulation of defective mitochondria in these mutants lead to increased Relish signalling, caused intestinal dysfunction and resulted in cell death in the midgut. Enhanced Relish signalling also resulted in metabolic alterations in pink1-mutant ies. These alterations are characterized by the accumulation of triglycerides (TAGs) due to the Relish-dependent regulation of lipid catabolism 5 and the failure of beta oxidation 11 . The genetic suppression of Relish or Eya, a Relishbinding protein involved in the sensing of cytosolic DNA, suppressed neurodegeneration in pink1-mutant ies. To understand the links between intestinal dysfunction and neurodegeneration, we subsequently investigated whether intestinal dysfunction can prime neurodegeneration and found that the prevention of intestinal dysfunction through either the suppression of Relish expression or the blockage of cell death in the midgut of pink1-mutant ies is su cient to suppress the central nervous system (CNS) phenotypes of these ies. We conclude that the CNS defects in pink1-mutant ies result from a non-cell autonomous signalling pathway induced by mitochondrial toxicity acting between the intestine and the brain.

Results
Identi cation of a Relish transcriptional signature in pink1-mutant ies.
We previously showed that mitochondrial defects in pink1-mutant ies lead to the upregulation of both nucleotide metabolism and immune response genes 11 . Here, we rst explored the mechanism underlying the activation of the innate immune response pathways associated with mitochondrial dysfunction in pink1-mutant ies. We analysed innate immunity-related transcripts and proteins in pink1-mutant ies through an in silico approach (Fig. 1A) and detected 42 upregulated transcripts that matched a curated list of innate immunity-related genes in ies (Fig. 1B and Supplementary Table 1). Because pink1-mutant ies exhibit a global shutdown of protein synthesis 12 , we also measured the levels of individual proteins in adult ies through quantitative proteomics. We detected the upregulation of nine proteins belonging to the innate immunity pathways in pink1-mutant ies (Fig. 1B and Supplementary Table 2). The transcriptional control of the innate immune response in Drosophila is mediated by signalling cascades that regulate the NF-kB-like transcription factors Dif, Dorsal and Relish (Rel) (reviewed in 13 ). To identify the upstream regulators of the innate immunity signature present in pink1-mutant ies, we used iRegulon, a tool used for the reverse-engineering of transcriptional networks 14 . This analysis identi ed Rel as the top upstream regulator of the innate immunity signature in pink1-mutant ies (Fig. 1B and  Supplementary Table 3).
A relish mutation suppresses the neuronal defects in pink1-mutant ies.
In ies, mutations in pink1 affect neuronal function, which leads to disruption of circadian rhythms in young adults 15 by preventing the secretion of neuropeptides 16 and the selective loss of dopaminergic neurons in the protocerebral posterior lateral 1 (PPL1) cluster in aged ies 17 . We subsequently tested whether mutation of the Relish gene in Rel E20 /+ ies 18 affected the neuronal phenotype of pink1-mutant ies. Speci cally, we monitored the locomotor activity during the light-dark cycle (LD) for 7 days and con rmed that pink1-mutant ies show aberrant activity patterns ( Fig. 2A). As previously reported 19 , pink1-mutant ies exhibited a signi cantly longer rest (or inactivity) duration, which was correlated with lower activity levels. Interestingly, the comparison of pink1-mutant ies with pink1,RelE20/+ doublemutant ies revealed that the Relish mutation led to a signi cant rescue of the sleep-wake patterns observed in pink1-mutant ies toward those of control ies ( Fig. 2A, red and blue). Additionally, the presence of a Relish mutation in the pink1-mutant ies was su cient to rescue the loss of DA neurons (Fig. 2B).
The activity of Relish during infection is negatively regulated by calcineurins and protein phosphatases. Calcineurins can be targeted by immunosuppressants, such as tacrolimus, which is used in human organ transplantation to lower the risk of rejection. Calcineurins are present in Drosophila 20 , and we thus tested whether pharmacological intervention with tacrolimus could suppress dopaminergic neurodegeneration in pink1-mutant ies. The exposure of these mutants to a diet supplemented with tacrolimus prevented the selective loss of dopaminergic neurons in the PPL1 cluster (Fig. 2C). Collectively, these ndings show that suppressing innate immunity pathways by manipulating Relish signalling is neuroprotective in a model of pink1 mutation-induced mitochondrial dysfunction.
Relish can induce immune responses to pathogens by inducing the transcription of antimicrobial peptides such as Attacin 21 . We subsequently monitored the activation of Relish by expressing a GFP transgene fused to the Attacin-A promoter (Att-GFP) 22 and detected GFP uorescence in several regions of the pink1-mutant ies, including their abdominal region (Fig. 3A). A more detailed analysis of the gut showed an increase in GFP expression in the gut enterocytes of pink1-mutant ies (Fig. 3B), which indicated the activation of Relish in the gut. Relish activation involves cleavage of an inhibitory domain on the full-length protein and translocation of the cleaved domain, Rel-68, to the cell nucleus (reviewed in 4 ). We subsequently monitored the levels of cytosolic Relish and found lower levels of the full-length protein (Fig. 3C) and increased levels of the Relish transcript ( Fig. 3D) in the pink1-mutant ies. Taken together, these results suggest that mitochondrial dysfunction in pink1-mutant ies is associated with Relish activation.
The increase in the expression of immunity-related transcripts is tightly linked to intestinal barrier dysfunction in Drosophila 23 . We subsequently performed a Smurf assay, which is a non-invasive method for determining the intestinal integrity in adult ies. The Smurf assay showed that pink1-mutant ies exhibit a compromised intestinal barrier that can partially be rescued by a mutation in Relish (Fig. 3E).
In Drosophila, the fat body, which is a lipid storage reservoir, and the gut form the primary immune organs. To visualize the gut and fat body in pink1-mutant ies, we labelled these tissues with phalloidin (to mark the gut) and BODIPY, a uorescent lipophilic dye that stains neutral lipids. A confocal microscopy analysis of pink1-mutant ies showed a ring of visceral fat (fat 'doughnut') surrounding the posterior end of the posterior midgut (anterior of the Malpighian tubule junction) (Fig. 4A). Fat body cells migrate to sites of tissue damage, where they clear wound-related cell debris and upregulate antimicrobial peptides (AMPs) 22 . Given their role in cell repair and renewal, we subsequently assessed whether the recruitment of fat body cells to the midgut was associated with damage to the midgut. We therefore assessed the degree of apoptotic cell death in the midgut of pink1-mutant ies and found increased levels of the active Drosophila caspase Dcp-1, an apoptosis effector (Figs. 4B and 4D), which indicated that pink1-mutant ies exhibit increased intestinal damage. Damage to the intestine also results in a robust proliferative response by intestinal stem cells (ISCs) (reviewed in 24 ). We also monitored the levels of ISC proliferation using a GFP reporter for Escargot (Esg), which is expressed in these cells 25  In Drosophila, the Eya protein acts in a cascade that senses undigested cytosolic DNA and activates the immune response. Eya binds to the inhibitor of nuclear factor kappa-B kinase subunit beta (IKKβ) 10 and full-length Relish 10,28 . Because neuronal dysfunction in pink1-mutant ies is suppressed by a Relish mutation, we subsequently tested whether eya can act upstream of Relish in response to naked mtDNA. We noted that the mRNA levels of Relish target genes were decreased in pink1-and-eya double-mutant ies (Fig. 5D). The eya mutation also rescued the defects in activity observed in pink1-mutant ies ( Fig. 5E, red and blue) and the loss of DA neurons (Fig. 5F). We conclude that the neurotoxic consequences of the activation of the immune response in pink1-mutant ies are linked to the presence of extramitochondrial DNA in the cytosol.
Relish signalling induces a starvation signature in pink1-mutant ies.
Drosophila pink1 mutants exhibit alterations in their circadian clock 15 , and here, we found that these ies also present defects in their gastrointestinal tract. Takeout is a Drosophila clock-controlled hormone that is primarily involved in feeding behaviour 29 and is strongly expressed in the crop, which results in dilatation of the oesophagus that serves as a food reservoir (Fig. 6A), the fat body and the antennae of male ies. Takeout expression is also induced upon starvation 29 . We detected an increase in the level of Takeout in pink1-mutant ies ( Fig. 6B and Supplementary Table 2). Furthermore, the measurement of the transcript levels of this circadian output hormone revealed that this hormone was upregulated in pink1mutant ies and that this upregulation was partially suppressed in pink1, Rel E20 /+ double-mutant ies (Fig. 6C). The Drosophila genome encodes eight different insulin-like peptides (DILPs), and four of these peptides (DILP1, DILP2, DILP3 and DILP5) are functionally similar to human insulin and are produced by neuronal insulin-producing cells (IPCs) in the y brain (Blue, Fig. 6A). Starvation, or low nutrient levels, decreases the release of DILPs by IPCs (reviewed in 30 ). The increase in Takeout, a starvation marker, together with the report that another clock-regulated hormone, insulin-like peptide 2 (DILP2), is arrested in the cell bodies of IPCs in pink1-mutant ies 16 led us to investigate whether these ies suffer from disruptions in metabolic and energy homeostasis. First, we focused on insulin, which is known to regulate dietary metabolism 31 . A confocal analysis of an epitope tagged DILP2 32 showed that pink1-mutant ies have higher levels of DILP2 in IPCs (Fig. 6D), which con rmed previous observations 16 . We then measured the total content of DILP2 peptide through an enzyme-linked immunosorbent assay (ELISA)based assay 32 and found that the levels of DILP2 in aged pink1-mutant ies were lower than those in the controls (Fig. 6E). Systemic repression of insulin signalling leads to a FOXO-dependent activation of NFκB-Relish signalling, which results in the induction of triglyceride (TAG) metabolism upon starvation 5 .
Fasting induces the mobilization of stored TAGs from lipid droplets that are used by other tissues and organs as a source of energy. We subsequently measured the TAGs in control and pink1-mutant ies and found signi cant accumulation of TAGs in both young and aged pink1B9 ies (Fig. 6F), and Relish mutation restored the TAG levels in old ies to the normal levels ( Fig. 6H), in accordance with previous ndings 5,33 . Taken together, these results link intestinal dysfunction in pink1-mutant ies to an imbalance in hormones related to feeding and changes in energy storage in adult ies.

The suppression of intestinal toxicity in pink1-mutant ies is neuroprotective
We previously showed that degeneration of the indirect ight muscles of pink1-mutant ies occurs in a non-cell autonomous manner through signalling between neurons and muscle cells 11 . Recent ndings in models of PD have shown that α-synuclein pathology occurs via inter-organ communication between the gastrointestinal tract and the brain in mice (reviewed in 34 ). Similar to those of mice, the central nervous system and gut of ies are interconnected. The Drosophila brain is connected to the midgut by neurons that produce insulin-like peptide 7 (ILP7) (Fig. 6A). The cell bodies of these neurons are located in the abdominal ganglion, and these cells innervate the midgut/hindgut junction and the rectal ampulla (reviewed in 35 ).
To test whether gut-brain communication could cause neurotoxicity in pink1-mutant ies, we rst suppressed Relish expression in the midgut by RNAi using the NP3084 midgut-speci c 36,37 Gal4 driver. This downregulation of Relish in the midgut decreased the overall levels of TAGs in pink1-mutant ies (Fig. 7A) and increased the level of fatty acid oxidation (Fig. 7B). These effects improved the mitochondrial function in the brains of pink1-mutant ies (Fig. 7C) and suppressed the inactivity defects (Fig. 7D) and the loss of DA neurons (Fig. 7E). We reason that the neurotoxic consequences of the activation of the immune response in pink1-mutant ies are linked to a gut-brain communication mechanism.
We subsequently tested whether the blockage of cell death in the intestine of pink1-mutant ies was su cient for the suppression of neurotoxicity. We targeted the expression of either Buffy, a Drosophila Bcl-2-like protein with anti-apoptotic activity 38 , or re-expressed Pink1 in the gut of pink1-mutant ies using the midgut driver. We found that the expression of either Buffy or Pink1 decreased the number of DCP1-positive cells (Figs. 8A and 8B) and prevented the loss of DA neurons in pink1-mutant ies (Fig. 8C). We thus conclude that the suppression of intestinal dysfunction in pink1-mutant ies is su cient for rescuing neurotoxicity.

Discussion
Mitochondria tune their performance to adjust their energy output to the requirements of individual cells.
To achieve this tuning, mitochondria communicate with the cell nucleus using the retrograde response 39 , which modi es the ow of information in cells by altering the transcriptional control of cellular functions. Here, we found that Relish signalling serves as a retrograde response pathway that is activated by mitochondria and has neurotoxic consequences. Relish, the Drosophila orthologue of NF-kB in mammals, controls the expression of several immunity genes. Recent studies in humans and mice have shown that PINK1 plays a role in restraining innate immunity and that in ammation plays a positive role in PD 40 .
Here, we extend these observations and demonstrate that in ies, this in ammatory response is triggered in the intestine and communicated to the central nervous system. These conclusions build on the increasing body of evidence obtained from models of α-synuclein pathology (reviewed in 34 ) that show a role for the gut-brain axis in the aetiology of PD.
We detected increased levels of enterocyte cell death in the intestines of pink1-mutant ies (Fig. 4). In Drosophila, Relish drives the removal of cells that are perceived un t via apoptotic cell death 41 , and we reason that the release of DNA from defective mitochondria in pink1-mutant ies labels cells as un t and prone to Relish-mediated apoptosis. It is also possible that a defective intestinal barrier in pink1-mutant ies allows the invasion of gut bacteria in the intestine and activates an in ammatory response.
In Pink1-knockout mice, mitochondrial dysfunction induces an in ammatory response via the cGAS-STING pathway that senses cytosolic DNA and acts upstream of the Relish orthologue NF-kB 40 . In Drosophila, dmSTING functions downstream of Relish to control viral infection 42 , and a previous study showed that its loss of function does not rescue the pink1-mutant phenotype 43 . Our data (Fig. 5) suggest that eya can act upstream of Relish to sense cytosolic DNA released by defective mitochondria in pink1mutant ies.
The defects in insulin signalling observed in pink1 mutants result from the abnormal tra cking of lipids between mitochondria and the endoplasmic reticulum, which in turn affects the formation of lipid vesicles and the release of neuropeptides such as DILPs 16 . These defects in lipid tra cking arise from the increased contacts between mitochondria and the endoplasmic reticulum observed in pink1-mutant ies 12,16 . Although partial suppression of Relish rescued the lipid defects in pink1 B9 ies (Fig. 6H), it did not rescue the release of DILP2 (Fig. 6G), con rming previous reports that insulin signalling acts upstream of Relish 44 .
Low levels of insulin are often associated with nutrient de cits and starvation, as indicated by an increased expression of Takeout (Figs. 6B and 6C) 29 , which results in reduced inhibition of FOXO by Akt and thus promotion of a stress response 45 . However, Relish can shape metabolic adaptation by attenuating Foxo-mediated lipolysis 5 , which leads to the accumulation of lipids 46 . Therefore, the sustained activation of Relish in pink1-mutant ies is likely to lead to the accumulation of lipids by blocking FOXO-mediated lipolysis.
We observed an accumulation of TAGs in pink1-mutant ies. Mitochondrial impairment has been shown to result in TAG accumulation 47 . We previously reported that dicarboxylate fatty acids are increased in pink1 mutants, and this increase is likely linked to defects in fatty acid beta oxidation 11 . Therefore, the accumulation of TAGs in these mutants might result from the failure of fatty acid beta oxidation due to mitochondrial impairment.
In rodents, the gut-brain transmission of PD pathology by α-synuclein occurs via the vagus nerve in mice 34 . Both retrograde and anterograde vagal transport act to promote the bidirectional propagation of αsynuclein toxicity to the brain and other organs in rats 48 .
Our results (Figs. 7 and 8) show that a gut-brain pathway is involved in neurotoxicity in a Drosophila model of PD. However, unlike the results obtained in α-synuclein models, our model showed that this pathway does not involve the transport of a toxic protein but rather a cell intrinsic signal induced by defective mitochondria.
In Drosophila, neurons with cell bodies in the posterior segments of the abdominal ganglion of the ventral nerve cord (VNC) send axons that reach the posterior portion of the midgut (reviewed in 35 ), which is the region where we detected intestinal dysfunction in pink1-mutant ies. Because this link between the intestine and the CNS via the VNC is established by ILP7 neurons (Fig. 6A), these neurons, similar to the vagus nerve in rodents, might mediate toxicity between the midgut and brain in pink1-mutant ies. It would be interesting to test whether silencing the activity of these neurons would affect CNS toxicity in pink1-mutant ies.
Alternatively, the communication of toxicity from the intestine to the CNS in pink1 mutants could involve an indirect (humoural) mechanism. A previous study showed that AMPs induced by Relish directly induce mitochondrial depolarization 49 . AMPs in the intestine of pink1-mutant ies might act as a humoural signal to induce the loss of mitochondrial membrane potential in the CNS of these ies.
Our data add to an increasing body of evidence showing that signals from the gut can modulate brain activity. More speci cally, our results suggest that mitochondrial toxicity in the gut can lead to adverse effects in the brain and that therapeutic interventions aiming to decrease gut toxicity might be a viable approach for restoring brain health in diseases caused by mitochondrial compromise.

Genetics and Drosophila strains
The y stocks and crosses were maintained on standard cornmeal agar media at 25°C. The strains used were the following: pink1 B9, white 1118 and w; elav>GAL4, which were previously described (Tu et  To distinguish pink1 B9 ies from ies with X-nondisjunction, we crossed pink1 B9 females to the Canton-S wild-type strain backcrossed to w 1118 ; thus, ies with nondisjunction have red eyes. This strategy was used for all crosses with mutants (i.e., Rel E20 and eya 2 ). For all other crosses, the ies were crossed to males carrying the FM7 balancer on the rst chromosome, i.e., pink1 B9 ;NP3084Gal4 x Fm7a; UAS-RelKK. All experiments involving adult ies were performed with males aged 3 to 5 days unless otherwise stated.

Microarray acquisition and analysis
RNA was obtained from the heads of male adult ies (six samples in total, three replicates of each genotype). The RNA quality was con rmed using an Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA). Detailed descriptions of the experimental protocols and raw data have been deposited in ArrayExpress under accession E-MTAB-6210. Differential expression was analysed using Partek Genomics Suite (Partek Inc., MO, USA) with an ANOVA model.
TMT labelling was performed according to the manufacturer's recommended protocol (https://www.thermo sher.com/order/catalogue/product/90110). One hundred micrograms of each digested protein sample was labelled individually with each of the 10 TMT tags. After labelling, the samples were combined, cleaned on a Sep-Pak C18 cartridge, dried and dissolved in 20 mM ammonium formate (pH 10). TMT peptide fractionation was performed using an Acquity ethylene-bridged hybrid C18 UPLC column (Waters; 2.1 mm i.d. x 150 mm, particle size of 1.7 µm). Dried fractions were separated using the LC-MS/MS method as detailed below. The fractions were combined into pairs (i.e., the rst fraction with the middle fraction) and analysed by LC-MS/MS using a Dionex Ultimate 3000 RSLC nanoUPLC (Thermo Fisher Scienti c Inc, Waltham, MA, USA) system and a Lumos Orbitrap mass spectrometer (Thermo Fisher Scienti c Inc, Waltham, MA, USA).

Data analysis
To explore the transcriptional changes in genes involved in the Drosophila immune response, transcripts with fold-changes (FCs) ≥ 2 in pink1-mutant ies were cross-checked with a list of Drosophila genes potentially involved in the immune response, which was made available as a resource by the Lemaitre group (lemaitrelab.ep .ch). The transcripts with FCs ≥ 2 in pink1-mutant ies annotated to this list were then analysed using the iRegulon algorithm 14 in Cytoscape (v3.5.1).
For proteomics analysis, the raw data les were processed using Proteome Discoverer v2.1 (Thermo Fisher Scienti c) and Mascot (Matrix Science) v2.6. The data were aligned with the UniProt data from Pseudomonas aeruginosa (5584 sequences), which is the common repository of adventitious proteins (cRAP, version 1.0). All comparative analyses were performed with the R statistical language. The R package MSnbase 51 was used for the processing of proteomics data. Brie y, this process entailed the removal of missing values (instances where a protein was identi ed but not quanti ed in all channels were rejected from further analysis), log2-transformation of the raw data, and subsequent sample normalization utilizing the 'diff.median' method in MSnbase (this translates all samples columns such that they all match the grand median). The differential abundances of the proteins were evaluated using the limma package, and the differences in protein abundances were statistically analysed using Student's t-test with their variances moderated by the empirical Bayes method in limma. The p-values were adjusted for multiple testing using the Benjamini Hochberg method 52 .

Locomotor assays
Three-to four-day-old males were individually loaded into Drosophila Activity Monitors (DAM5) within 8 × 65-mm glass Pyrex tubes (Trikinetics, Waltham, MA, USA) containing normal y food. The ies were maintained at 25°C under a 12-hour light:12-hour dark (LD) cycle for at least 8 days. Sleep and activity data were analysed using the Sleep and Circadian Analysis MATLAB Program (SCAMP) developed by the Gri th lab 53 . The analyses were performed for 7 days starting at the rst ZT0 to allow acclimation. At least 16 ies of each genotype were used.

Assay of the integrity of the intestinal barrier
We measured the integrity/functionality of the intestinal barrier by detecting the presence of a nonabsorbable blue food dye outside the digestive tract after feeding 54 . The ies were transferred to food containing 2.5% w/v Erioglaucine (FD&C Blue dye1-SIGMA) overnight 55 . Afterward, the ies were allowed to eat normal food for an additional 24 hours and then retransferred to dye-containing food for an additional 24 hours. The ies were scored after being fed normal food for 48 hours. Notably, in the analysis of Smurf ies fed normal food for 48 hours, a stained abdomen served as an indication of GI dysfunction, and we did not score different degrees of "smurfness". A pairwise test followed by a stack of p-values with an FDR of 10% was used to detect the signi cance of the differences among genotypes.

Drug treatments
Tacrolimus (FK-506 monohydrate, Sigma, F4679) was incorporated into the y food to a nal concentration of 5 mM. Flies treated with tacrolimus were transferred to drug-containing food up to 24 hours after hatching.

Protein extraction and western blotting
Ten to fteen guts were dissected in PBS and maintained on ice until ready to be processed. Protein extraction was performed using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo Scienti c, 78833) following the manufacturer's instructions. The samples were separated using BioRad Precast gels and wet-blotted onto nitrocellulose.

Antibodies and dyes
The primary antibodies and dyes employed in this study were anti-α-tubulin (CST, 2125), anti-cleaved

RNA extraction and quantitative real-time RT-PCR
Total RNA was extracted from 10-15 freshly dissected guts using TRIzol (Ambion) and quanti ed by spectrophotometric analysis (Nanodrop, Thermo Scienti c). Quantitative real-time PCR with reverse transcription (qRT-PCR) was performed with a real-time cycler (Applied Biosystems 7500 Fast Real-Time PCR Systems) using the SensiFAST SYBR Lo-ROX One-Step Kit (Bioline). Gene-speci c primers were obtained from Sigma, and rp49 was used as a housekeeping gene.
Immuno uorescence and confocal microscopy For brain imaging, the ies were xed overnight at 4°C in 4% PFA/1% Triton/PBS. The brains were subsequently dissected in ice-cold PBS and blocked in 10% normal goat serum PBS/0.5% Triton overnight. Primary staining was performed at 4°C for 3 days, whereas the samples were incubated with the secondary antibodies for 2 hours at room temperature.
For intestinal imaging, the guts were dissected from live ies in PBS and xed in 4% PFA for 1 hour on ice.
The staining protocol was identical to that used for the brain samples.
For DCP-1 staining, the guts were xed in heptane-methanol xative. The samples were mounted in Vectashield (Vector Laboratories), and uorescence images were acquired with a Zeiss LSM880 confocal microscope.

Microscopy-based assessment of mitochondrial function and morphology
Measurements of the Δψm in y brains were performed as previously described 11 . Brie y, y brains were loaded for 40 minutes at room temperature with 40 nM TMRM in loading buffer (10 mM HEPES pH 7.35, 156 mM NaCl, 3 mM KCl, 2 mM MgSO 4 , 1.25 mM KH 2 PO 4 , 2 mM CaCl 2 , and 10 mM glucose), and the dye was present during the experiment. In these experiments, TMRM was used in the redistribution mode for the assessment of Δψm, and therefore, a reduction in the TMRM uorescence represents mitochondrial depolarization. Confocal images were obtained using a Zeiss LSM 880 confocal microscope equipped with a 40x oil immersion objective. The illumination intensity was kept to a minimum (0.1-0.2% of the laser output) to avoid phototoxicity, and the pinhole was set to yield an optical slice of 2 μm. The uorescence was quanti ed by exciting TMRM using the 565-nm laser and measured above 580 nm. Zstacks of ve 300-μm 2 elds were acquired from each brain, and the mean maximal uorescence intensity of each group was measured.

Analysis of dopaminergic neurons
The brains from 25-day-old ies were dissected and stained with anti-tyrosine hydroxylase antibody (Immunostar) as previously described 56 . The brains were positioned in PBS + 0.1% Triton in a coverslip clamp chamber (ALA Scienti c Instruments Inc., NY, USA) using a harp composed of platinum wire and nylon string and imaged by confocal microscopy. The numbers of tyrosine hydroxylase-positive neurons in the PPL1 cluster was determined for each brain hemisphere. The data acquired for the assessment of each genotype were obtained as a single experimental set prior to statistical analysis.

Metabolic assays
The assessment of DILP2 was performed by ELISA, whereas the levels of TAGs and beta oxidation were assessed through colorimetric assays using 96-well microtiter plates and an In nite M200Pro multifunction reader (TECAN, Mannedorf, Switzerland).
For the metabolic assays, groups consisting of a maximum of 20 male ies were placed on normal foodcontaining vials, and the food was changed every 2 days until the desired age was reached, i.e., either 3 or 30 days, and maintained in an 12:12 LD at 25°C. Day 0 was considered the day when the ies emerged from the pupal case. At the de ned ageing stage, the ies were transferred into vials containing cotton soaked with water for 2 hours to minimize food contamination from ZT 2 to ZT 4. CO 2 -anaesthetized ies were then divided into groups of ve ies and ash frozen for subsequent analysis. The TAG and glycogen assays were essentially performed as previously described 57 . Brie y, for TAG analysis, ve adult ies were homogenized in 100 µL of PBS + 0.05% Tween-20 (PBSTw) for 60 seconds on ice and immediately incubated at 70°C for 10 minutes for the inactivation of endogenous enzymatic activity.
Forty microlitres of the y samples and glycerol standards (SIGMA, G7793) were incubated together with either 40 µL of PBST (for free glycerol measurements) or 40 µL of TAG reagent (SIGMA T2449, for TAG measurements) at 37°C for 60 minutes.
After 3 minutes of centrifugation at full speed, 30 µl of each sample (two technical replicates/biological sample) was transferred into a clear-bottom plate together with 100 µL of free glycerol reagent (SIGMA, F6428) at 37°C for 5 minutes.
For insulin ELISA, the ies were maintained and aged as for the TAG assay with the modi cation that the ies were not starved for 2 hours but rather collected at ZT 2. The ELISA was performed as previously described 32 with the modi cation that 5 µL of anti-HA-HRP antibody diluted 1:350 in PBS was added to 50 µL of protein samples as described by 58 . Subsequently, the TAG and DILP2 absorbances were divided by the protein concentration of the respective sample, which was measured by the Bradford assay. The results were normalized to the control group by dividing the average values 59 .
Similarly, for beta oxidation analysis, 10 ies of the desired age were collected and maintained on ice following the manufacturer's instructions.
For beta oxidation analysis, the ies were maintained and aged as described above with no starvation step and collected at ZT 2. Brie y, 15 ies (for each biological replicate) were anaesthetized, transferred to Eppendorf tubes, weighed and maintained on ice. Approximately 10 mg of tissue was used for each replicate. The ies were subsequently homogenized in 200 µL of provided lysis buffer, and the lysate was clari ed by centrifugation at 4°C. The Bradford assay was used for quanti cation of the protein concentration, and all the samples were normalized to 2 mg/mL. The assay was then performed based on the protocol suggested by the manufacturer (BMR E-141). Incubation at 37°C was performed for 1 hour.

Statistical analyses
The statistical analyses were performed using GraphPad Prism 8 (www.graphpad.com). The data are presented as the mean values, and the error bars indicate the ±SDs. In violin plots, the solid line represents the median, whereas the dotted lines represent the quartiles. The number of biological replicates per experimental variable (n) is indicated in either the respective gure or the gure legend. The signi cance is indicated as follows: * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

Digital image processing
Fluorescence and western blot images were acquired as uncompressed bitmapped digital data (TIFF format) and processed using Adobe Photoshop with established scienti c imaging work ows 60 . To visualize the pixel intensity, confocal images acquired with identical settings were processed using a 5tone heat map (f.64 Academy) in Photoshop.