Tachykinin-related peptide signaling and its role in metabolic stress in Drosophila

Sammanfattning: Tachykinins (TKs) constitute a highly conserved family of multifunctional neuropeptides that are known to be involved in a multitude of functions in mammals. Peptides that are presumed ancestrally related to tachykinins, so called Tachykinin-related peptides (TKRP) have been identified in invertebrates.Little is known about the in vivo actions of TKRPs in invertebrates. Therefore, we decided to study the TKRP signaling in the Drosophila melanogaster. Drosophila has been shown to have a tachykinin system with multiple peptides encoded on a single gene (Tk) and two G-protein coupled receptors (GPCRs), designated DTKR and NKD. The two GPCRs were further characterized in this thesis with native DTKs.We raised several antibodies for immunocytochemical detection of DTKR and NKD to investigate the distribution of the two receptors. We found that the two GPCRs are differentially distributed and when combined cover all regions of the DTK distribution.To analyze receptor function in vivo we used the GAL4-UAS system to over express DTKR and NKD with different cell specific GAL4 drivers, likely to shed light on metabolic stress responses. We also tested the effect of a DTK RNA interference (Tk-KO) transgene, in, which levels of DTK transcript was decreased by up to 95%. These flies were tested for different parameters like trehalose and lipid levels, longevity, and locomotor activity levels at starvation. We found that the Tk-KO flies displayed a prolonged lifespan and no detectable decrease trehalose. We also showed that over expression of the two GPCRs elicits different responses depending on the cell type they are expressed in. Over expression of NKD in AKH producing cells is likely to cause a release of adipokinetic hormone (AKH) while DTKR appears to have an inhibitory action on release of Drosophila insulin-like peptides (DILPs) when expressed on DILP2 producing cells.It has been previously shown in the locust that TKRPs are released into the haemolymph from the midgut upon starvation. We therefore used Gal4s specifically directed to the two different cells types (principal and stellate cells) of the Malpighian tubules to drive DTKR and NKD. Only when expressing the DTKR receptor in the principal cells did we see an altered phenotype during nutritional and desiccative stress. We found that over expression of DTKR produced an abbreviated lifespan. We also tested the Tk-KO flies and found that they exhibited under desiccative stress. Finally, we expressed a DTKR-RNAi construct in the both Malpighian tubules cells type and found that in both cell types the receptor knockdown produced an extended lifespan compared to controls. These data suggests that since only substances in the circulation can activate receptors on the Malpighian tubules DTKs are likely to be released into the circulation upon starvation. In summary our data indicate that DTKs and the two GPCRs are involved in signalling during metabolic stress.