Negative Feedback Mechanisms Regulating Neurotransmitter Release at the Drosophila Neuromuscular Junction

摘要

Homeostasis is an indispensable phenomenon in the maintenance of living organisms. Genetic defects which disrupt negative feedback processes can impact homeostatic regulation, potentially resulting in disease. To uncover the molecular mechanisms governing these and other diseases potentially related to defective homeostasis, I used the Drosophila neuromuscular junction as a model system. I characterized two potential mechanisms that regulate homeostasis within the nervous system. First, in Drosophila larval motor neurons, ligand activation of Drosophila metabotropic glutamate receptor A (DmGluRA) mediates a Phosphoinositide 3-kinase (PI3K)-dependent downregulation of neuronal activity, but the mechanism by which mGluR activates PI3K remains incompletely understood. Here, I identified Ca 2+ /Calmodulin-dependant protein kinase II (CaMKII) and the Focal adhesion kinase (DFak) as critical intermediates in the DmGluRA-dependent activation of PI3K at Drosophila motor nerve terminals. I found that transgene-induced CaMKII inhibition or the DFak CG1 null mutation each block the ability of glutamate application to activate PI3K in larval motor nerve terminals, whereas transgene-induced CaMKII activation increases PI3K activity in motor nerve terminals in a DFak-dependent manner, even in the absence of glutamate application. I conclude that the activation of PI3K by DmGluRA is mediated by CaMKII and DFak. Second, I observed that Push, a putative E3-ubiquitin ligase and Ca 2+ /Calmodulin binding protein, regulates both neurotransmitter release and retrograde signaling in the Drosophila neuromuscular junction. I found that RNAi-mediated Push inhibition in the neuron increases but, in the muscle decreases, neurotransmitter release. Similar results were obtained from RNAi knock down of PLCβ and IP3R, which mediates Ca 2+ release from the endoplasmic reticulum. I conclude that Push mediation of the ubiquitin proteasome system may be important in the regulation of PLCβ/IP3R-mediated intracellular Ca 2+ release, and that this Ca 2+ release in the neuron inhibits neurotransmitter release, but in the muscle activates neurotransmitter release via a retrograde signal.