Insulin and IGF signaling are critical to numerous developmental and physiological processes, with perturbations being pathognomonic of various diseases, including diabetes. Although the functional roles of the respective signaling pathways have been extensively studied, the control of insulin production and release is only partially understood. Herein, we show that in Drosophila expression of insulin-like peptides is regulated by neprilysin activity. Concomitant phenotypes of altered neprilysin expression included impaired food intake, reduced body size, and characteristic changes in the metabolite composition. Ectopic expression of a catalytically inactive mutant did not elicit any of the phenotypes, which confirms abnormal peptide hydrolysis as a causative factor. A screen for corresponding substrates of the neprilysin identified distinct peptides that regulate insulin-like peptide expression, feeding behavior, or both. The high functional conservation of neprilysins and their substrates renders the characterized principles applicable to numerous species, including higher eukaryotes and humans.

Neuropeptides and peptide hormones serve as critical regulators of numerous biological processes, including development, growth, reproduction, physiology, and behaviour. In mammals, peptidergic regulatory systems are complex and often involve multiple peptides that act at different levels and relay to different receptors. To improve the mechanistic understanding of such complex systems, invertebrate models in which evolutionarily conserved peptides and receptors regulate similar biological processes but in a less complex manner have emerged as highly valuable. Drosophila melanogaster represents a favoured model for the characterisation of novel peptidergic signalling events and for evaluating the relevance of those events in vivo. In the present study, we analysed a set of neuropeptides and peptide hormones for their ability to modulate cardiac function in semi-intact larval Drosophila melanogaster. We identified numerous peptides that significantly affected heart parameters such as heart rate, systolic and diastolic interval, rhythmicity, and contractility. Thus, peptidergic regulation of the Drosophila heart is not restricted to chronotropic adaptation but also includes inotropic modulation. By specifically interfering with the expression of corresponding peptides in transgenic animals, we assessed the in vivo relevance of the respective peptidergic regulation. Based on the functional conservation of certain peptides throughout the animal kingdom, the identified cardiomodulatory activities may be relevant not only to proper heart function in Drosophila, but also to corresponding processes in vertebrates, including humans.

Drosophila nephrocytes are specialised cells that share critical functional, morphological, and molecular features with mammalian podocytes. Accordingly, nephrocytes represent a preferred invertebrate model for human glomerular disease. Here, we established a method for cell-specific isolation of the two types of Drosophila nephrocytes, garland and pericardial cells, from animals of different developmental stages and ages. Mass spectrometry-based proteomics and RNA-Seq-based transcriptomics were applied to characterise the proteome and transcriptome of the respective cells in an integrated and complementary manner. We observed characteristic changes in the proteome and transcriptome due to cellular ageing. Furthermore, functional enrichment analyses suggested that larval and adult nephrocytes, as well as garland and pericardial nephrocytes, fulfil distinct physiological functions. In addition, the pericardial nephrocytes were characterised by transcriptomic and proteomic profiles suggesting an atypical energy metabolism with very low oxidative phosphorylation rates. Moreover, the nephrocytes displayed typical signatures of extensive immune signalling and showed an active antimicrobial response to an infection. Factor-specific comparisons identified novel candidate proteins either expressed and secreted by the nephrocytes or sequestered by them. The data generated in this study represent a valuable basis for a more specific application of the Drosophila model in analysing renal cell function in health and disease.

Muscle contraction depends on strictly controlled Ca²⁺ transients within myocytes. A major player maintaining these transients is the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase, SERCA. Activity of SERCA is regulated by binding of micropeptides and impaired expression or function of these peptides results in cardiomyopathy. To date, it is not known how homeostasis or turnover of the micropeptides is regulated. Herein, we find that the Drosophila endopeptidase Neprilysin 4 hydrolyzes SERCA-inhibitory Sarcolamban peptides in membranes of the sarcoplasmic reticulum, thereby ensuring proper regulation of SERCA. Cleavage is necessary and sufficient to maintain homeostasis and function of the micropeptides. Analyses on human Neprilysin, sarcolipin, and ventricular cardiomyocytes indicates that the regulatory mechanism is evolutionarily conserved. By identifying a neprilysin as essential regulator of SERCA activity and Ca²⁺ homeostasis in cardiomyocytes, these data contribute to a more comprehensive understanding of the complex mechanisms that control muscle contraction and heart function in health and disease.

Arrhythmogenic right ventricular cardiomyopathy type 5 (ARVC-5) is a fully penetrant form of ARVC caused by the missense mutation in the gene TMEM43 p.S358L. Despite extensive research, the molecular basis underlying the detrimental effects of TMEM43 p.S358L still needs to be discovered. TMEM43 is a phylogenetically conserved protein. We previously analysed the Drosophila homologue (CG8111 or Dmel\Tmem43) to understand the protein's physiological relevance and the mutation p.S358L. Drosophila Tmem43 is localised at the ER/SR membrane and interacts with the outer mitochondrial membrane protein Porin/VDAC. This interaction is lost in a Tmem43p.S333L mutant that resembles the human p.S358L mutation. In addition, Tmem43p.S333L caused a breakdown in mitochondrial membrane potential and increased cellular reactive oxygen species, suggesting impaired mitochondrial function as a major pathomechanism. Complementary ultrastructural analyses revealed severe structural defects in the affected mitochondria, including degeneration of the organelles. Highly similar ultrastructural defects were observed in the human right ventricular myocardium of a TMEM43p.S358L trait carrier, suggesting a common molecular basis for the detrimental effects of the mutation in flies and humans. We propose that both the p.S358L mutation in humans and the p.S333L mutation in Drosophila impair TMEM43/VDAC interaction, which affects the stability of ER/SR-mitochondrial contact sites and, thus, proper mitochondrial function and oxidative phosphorylation rates. The consequential undersupply of ATP likely results in cardiac cell death and, ultimately, heart failure.

Appropriate cardiac performance depends on a tightly controlled handling of Ca²⁺ in a broad range of species, from invertebrates to mammals. The role of the Ca²⁺ ATPase, SERCA, in Ca²⁺ handling is pivotal, and its activity is regulated, inter alia, by interacting with distinct proteins. Herein, we give evidence that 4E binding protein (4E-BP) is a novel regulator of SERCA activity in Drosophila melanogaster during cardiac function. Flies over-expressing 4E-BP showed improved cardiac performance in young individuals associated with incremented SERCA activity. Moreover, we demonstrate that SERCA interacts with translation initiation factors eIF4E-1, eIF4E-2 and eIF4E-4 in a yeast two-hybrid assay. The specific identification of eIF4E-4 in cardiac tissue leads us to propose that the interaction of elF4E-4 with SERCA may be the basis of the cardiac effects observed in 4E-BP over-expressing flies associated with incremented SERCA activity.