Molecular mechanisms underlying temperature-dependent synaptic growth at the <em>Drosophila melanogaster</em> neuromuscular junction

Abstract

There is clear evidence that Earth's temperature is rising at an unprecedented rate. While consequences on ecosystems are being extensively studied, little is known about the consequences of temperature on the nervous system of ectothermic animals. Using the stereotyped synapse found at the <em>Drosophila</em> neuromuscular junction (NMJ), we asked how rearing temperature (15°C, 25°C, 29°C) can affect synaptic growth. We observed an increase in the number of synaptic boutons in animals reared at higher temperature. Indeed, animals reared at 29°C had a 100% increase in synaptic growth when compared to animals reared at 15°C. Interestingly, we found that from the two motor neurons that innervating the muscle, the number of boutons from the Is motor neuron increased with temperature while the boutons from the Ib motor neuron remained constant. This result indicates that motor neurons might be differentially sensitive to the changes in temperature.<br /> <br /> We looked for the molecular mechanisms that regulate temperature-dependent synaptic growth. We identified that autophagy was important for this regulation. Autophagy mutants showed a temperature-independent undergrowth, where there was a reduction in the Is boutons, while the Ib boutons remained constant through the rearing temperatures. In addition, with the use of Lysotracker and the p62 marker, we identified that the levels of autophagy changed at different rearing temperatures. Afterwards we found that the autophagy target, Highwire (Hiw), an E3 ubiquitin ligase, is a key regulator of temperature-dependent synaptic growth. Hiw is a negative regulator of synaptic growth, and its mutation induced a temperature-independent overgrowth phenotype. In addition, we found two important MAPKKK pathways that are important for regulation of synaptic growth at 15°C and 29°C. The first is the Wallenda-P38b pathway which is important for the addition of Is boutons of animals reared at 29°C. The second is the ASK1-JNK-c-Jun pathway, which is important for suppressing synaptic growth in animals reared at 15°C. Also, we found that constitutive activation of the insulin pathway in animals reared at 15°C increases the total number of synaptic boutons, but activation of the same pathway at 25°C does not affect synaptic growth. We hypothesize that temperature-dependent synaptic growth is regulated by global mechanisms like autophagy and the ubiquitin proteosome system that regulate the activation of stress response components, like the MAP kinase pathways. By changing the rearing temperature, we have identified novel roles for signaling pathways that were previously described to regulate synaptic growth in loss-of function or gain-of-function mutant backgrounds.