Iditarod, a Drosophila homolog of the Irisin precursor FNDC5, is critical for exercise performance and cardiac autophagy

Significance This study identifies Iditarod (Idit), a Drosophila gene that is similar to mammalian FNDC5/Irisin protein, as a regulator of autophagy, exercise performance, and cold resistance. Mammalian FNDC5/Irisin was previously shown to be implicated in exercise physiology. Our findings reveal that the role of Idit/Irisin/FNDC5 family proteins is conserved across animal species, including invertebrates. In flies, Idit deficiency led to impaired exercise endurance and cold tolerance, while Idit overexpression increased exercise endurance. Additionally, Idit was necessary for exercise-induced cardiac autophagy and stress resistance. This work suggests that the Idit/Irisin/FNDC5 family has ancient roles in autophagy, exercise physiology, and cold adaptation, providing insights into the conserved functions and mechanisms of these proteins across metazoan species.


Antibodies, Immunoblotting and Fluorescence Imaging
Rabbit anti-Idit antibodies were made as follows: Idit coding sequence was cloned into pGEX and transformed into E.coli BL21 strain.Insoluble GST-Idit proteins were purified from SDS-PAGE bands and injected into rabbits (Pocono farms, Inc.).Sera were subjected to affinity purification using PVDF-immobilized proteins.Atg1 and anti-Atg9 antibodies were formerly described (3).Atg13-GFP was detected using anti-GFP antibodies (Santa Cruz Biotechnology, sc-9996; 1:50).Anti-TUBA/a-tubulin (Sigma, T5168; 1:1000), anti-ACTA1/actin (DSHB, JLA20; 1:100), anti-GABARAP (anti-Atg8a; IHC -1:100; see (3) regarding cross-reactivity information), anti-Atp5a (Invitrogen; 1:500) antibody was used to detect endogenous levels of corresponding Drosophila proteins.For immunoblotting, cell and tissue lysates prepared in RIPA buffer (50 mM Tris-HCl pH 7.4; 150 mM NaCl; 1% deoxycholate Na; 1% NP-40; 0.1% SDS and complete protease inhibitor cocktail (Roche)) were boiled in 1X SDS sample buffer for 5 min at 95 o C. For detection of Idit, tissue lysates were processed in 1X Lammeli buffer (1610737, Bio-Rad) at 60 o C water bath for 5 min, chilled at ice for 5 min, and stored at 4 o C afterwards.The processed lysates were separated by SDS-PAGE, transferred to PVDF membranes and probed with primary antibodies and then with horseradish peroxidase-conjugated secondary antibodies.Chemiluminescence was detected using LAS4000 or AI680 (GE) systems.LysoTracker Red staining, acridine orange staining and live fluorescence imaging procedures are formerly described (3).Fluorescence images were quantified in ImageJ or Adobe Photoshop as previously performed (3)(4)(5) with some modifications.Heart tube area was measured and contrast was increased to identify Atg8-positive (red) pixels above a predetermined intensity threshold.The area of pixels above this threshold was measured, added together, and then calculated as a percent of total heart tube area.Statistical analysis of % heart area was performed with 2-way ANOVA with post-hoc Tukey test for multiple comparisons.

Chill Coma
The chill coma assay was performed as previously described (6).Five vials containing approximately 5-7 flies were transferred to fresh, empty vials and immediately submerged into ice for two hours to induce a chill coma.Vials were removed and flies were individually monitored for recovery, which was scored when an individual fly was standing upright.Unpaired t-test or one-way ANOVA was performed to measure the statistical difference between means.

Cardiac Pacing
Cardiac pacing was performed as previously described (5).Electrodes from a square wave stimulator were placed on each end of a modified microscope slide and secured with tinfoil.Conductive gel was placed on the foil on each end, leaving a 2-3 mm gap between the gel.Flies were placed between the gel with the head touching one side of the gel and the abdomen touching the other.Hearts were paced at 40V and 6 Hz for 30 seconds.Hearts were then visually scored for failure (i.e.not beating) immediately after pacing and results were presented as failure rate per total flies paced.Data was analyzed with a Chi-square test for significance.

Mammalian Cell Culture
Idit and mFNDC5 cDNA, obtained as described above, were subcloned into a pLU-CMV vector after being tagged with N-terminal or C-terminal Flag sequences.HEK293 cells (the 293A substrain from Invitrogen, tested negative for mycoplasma by PCR) were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) containing 10% fetal bovine serum (FBS, Sigma) and penicillin/streptomycin (Thermo Fisher Scientific) at 37°C in 5% CO2.For transient expression of proteins, HEK293 cells were transfected with purified plasmid constructs and polyethylenimine (PEI, Sigma).Cells were subjected to autophagic flux assays using Bafilomycin A1 (Sigma), as indicated in the figures, at 24 hr after transfection.

Quantitative RT-PCR
RNA was extracted from whole flies using TRIzol Reagent (Invitrogen) and was equalized to 30 ng/ul between groups.Relative mRNA abundance of Idit was measured with one-step RT-PCR.Reactions containing template RNA, SYBR Green (Applied Biosystems), MultiScribe reverse transcriptase (Applied Biosystems), RiboLock Rnase inhibitor (Thermo Scientific), primers, and water were run in a QuantStudio 3 RT-PCR System (Applied Biosystem).Comparative Ct (7) was used to quantitate relative abundance and fold change of Idit mRNA using Act5c as an internal control.Unpaired t-test was used to test for significance.Primers used are as follows: Act5c Forward: CGCAGAGCAAGCGTGGTA Act5c Reverse: GTGCCACACGCAGCTCAT Idit Forward: AGCAACGACATCGAGCTGAA Idit Reverse: CGCTAGCAGACTAGTCACGG

Figure S2 .
Figure S2.Idit silencing suppresses Atg1-Atg13-induced autophagy.(A and B) Developing eye discs from wandering-stage third instar larvae expressing indicated transgenes were analyzed by acridine orange (AO) staining (A) and Lysotracker staining (B) to visualize dying cells and autolysosomes, respectively.Brackets indicate the areas of differentiated ommatidia that express GMR-Gal4.Boxed areas are magnified in the panels below.(C) Developing eye discs from wandering-stage third instar larvae expressing indicated transgenes were analyzed by anti-Atg9 staining, which monitors trafficking of endosomes to autophagosomes.Boxed areas are magnified in the panels below.(D) Fluorescence staining of endogenous Atg8 (red) and visualization of Atg13conjugated GFP (green) in eye discs of indicated genotypes.Grey brackets: morphogenetic furrow region, Black and white brackets: anterior/early and posterior/late areas of post-morphogenetic furrow regions, respectively.(E and F) Quantification of Atg8 intensity (E) and Atg13-GFP ratio between anterior and posterior areas of post-morphogenetic furrow region (F).Error bars represent standard deviation.Asterisks indicate significance from Tukey's multiple comparison test; * p<0.05, ** p<0.01.

Figure S3 .Figure S4 .
Figure S3.Idit is the Drosophila homolog of Irisin/FNDC5.(A) Constraint-based Multiple Alignment of Idit and its related proteins.Red box indicates the putative Irisin cleavage motif, which is further magnified in (H).(B and C) Needleman-Wunsch global alignment of Idit and human FNDC5 (B) and subsequent dot plot analysis (C) that identifies putative domains including Irisin domain (yellow boxes), Irisin cleavage domain (black boxes, CL), transmembrane domain (red boxes, TM), Intracellular domain 1 (blue boxes, I1), and Intracellular domain 2 (green boxes, I2).Homology of each domain to human FNDC5 is assessed after Needleman-Wunsch alignment of each domain.Homology information for Irisin domain and TM domain can be found in Fig. 2B.Putative Irisin cleavage motif is highlighted in red circle (D) Domain structures of Idit-related proteins in Drosophila.Domain structure diagrams for Idit, Bt, Ptp99A, Lar, Ptp10D, Slo, and CG12541, which are the proteins that have substantial sequence homology to human Irisin/FNDC5, were obtained from Flybase and presented here.Protein domains were identified from Pfam.CG12541 lacks the fibronectin III domain (FN3_dom) that provides the Irisin homology.All unlabeled green and red domains correspond to FN3_dom.Except Idit and CG12541, all other proteins contain unrelated domains, such as immunoglobin, protein kinase, protein phosphatase and ion channel domains.The illustrations, while not accurately to scale, are approximately representative of the intended proportions of domains.Accurate protein lengths are indicated.(E and F) Phylogenetic tree of Idit and related proteins, constructed through Fast Minimum Evolution (E) and Cobalt tree dendrogram (F) implemented in NCBI.Total 13 proteins (shown in F) were analyzed; however, in Fast Minimum Evolution analysis, 6 proteins showed sequence differences of greater than 85%, so were excluded in the analysis.(G) Alphafold prediction of CG12541, which revealed no structures related to the Irisin domain.(H) Close-up view of the putative Irisin cleavage motif.