Synapses undergo many stresses and plastic changes throughout the life of an organism. Homeostatic mechanisms respond to these stresses and maintain synaptic activity within a physiologically favorable range. When faced with a reduction in postsynaptic glutamate receptor activity, the Drosophila neuromuscular junction (NMJ) homeostatically compensates by sending a retrograde signal to the presynaptic nerve. This signal triggers an increase in the number of synaptic vesicles released from the presynaptic terminal during an action potential.
One of the least well understood aspects of this process is how postsynaptic systems drive production of homeostatic retrograde signals. We have identified several factors that regulate homeostatic synaptic plasticity in the postsynaptic muscle through an RNAi- and electrophysiology-based screen. This screen revealed that C-terminal Src Kinase (Csk) and the fibroblast growth factor receptor (FGFR) Heartless (Htl) are required for homeostatic compensation at the NMJ. This led to the development of two projects, one focused on Csk and the other on Htl.
Work with Csk mutant alleles shows that Csk is required for the long-term maintenance of synaptic homeostasis, but not the rapid induction of this process. Csk phosphorylates and inactivates Src Family Kinases (SFKs), of which there are two in Drosophila: Src64B and Src42A. Overexpression and suppression experiments indicate that the homeostatic defects of Csk mutants are due to elevated SFK activity in the postsynaptic muscle. Immunostaining reveals that Csk mutants have altered NMJ levels and localization of the neural cell adhesion molecule (NCAM) ortholog Fasciclin II (FasII). Western analysis reveals that the change in FasII levels in Csk mutants results from upregulation of a previously unreported FasII isoform. We examined a potential role for FasII in homeostatic plasticity and found that increasing FasII levels partially impairs this process. Additionally, reducing FasII in a Csk mutant background restores homeostatic compensation, suggesting that Csk and FasII regulate homeostatic compensation through a common pathway.
We show that Htl/FGFR is required in the postsynaptic muscle for the maintenance, but not the induction, of homeostatic signaling. Htl is known to activate Src64B, and we show that Src64B is required for homeostasis in the postsynaptic muscle and link Src64B and Htl/FGFR signaling in the context of homeostatic compensation. We also determine that Htl signaling regulates homeostatic plasticity through activation of translation, likely through target of rapamycin (TOR). FasII has been implicated as a regulator of Htl activity in Drosophila, which is supported by our observation that FasII genetically interacts with Htl and Src64B during homeostatic compensation, connecting the homeostatic block of Csk mutants to Htl signaling.
Collectively, these data shed light on postsynaptic mechanisms that work in a single pathway to regulate the production of a homeostatic retrograde signal.