| Synapses are the foundation of the complex processing abilities of the nervous system. Synaptic function relies not only on the correct assembly of synaptic machinery, but also on the specialized neuronal morphology providing the framework for synaptic connections. To find novel mediators of synaptic development, we screened for mutants in synaptic morphology and growth at the Drosophila larval neuromuscular junction, a morphologically stereotyped synapse. Our screen uncovered a B' regulatory subunit of protein phosphatase 2A (PP2A), a heterotrimeric enzyme enriched in the nervous system, particularly during development. Despite PP2A's elevated expression in the immature nervous system, little is known about how PP2A regulates neuronal development. We therefore characterized the role of this Drosophila PP2A regulatory subunit, well-rounded ( wrd), in synaptic development. Genetic analysis of wrd mutants shows that wrd is required presynaptically for normal synaptic growth and morphology. Additionally, loss of wrd leads to decreased cytoskeletal stability, suggesting a mechanism by which wrd could regulate growth and morphology of the NMJ.; The requirement of a PP2A regulatory subunit for normal NMJ morphogenesis implies that PP2A activity influences synaptic development. To examine the synaptic role of PP2A activity, we inhibited neuronal PP2A via a dominant negative transgene of PP2A's catalytic subunit. Like loss of wrd, inhibition of PP2A disrupts the synaptic cytoskeleton. PP2A inhibition additionally disrupts the characteristic apposition between presynaptic release sites and postsynaptic receptors, an arrangement critical to synaptic transmission. Under reduced PP2A activity, numerous receptors at the NMJ lack a complementary pre-synaptic active zone. Younger larvae demonstrate relatively fewer unapposed glutamate receptors, consistent with a role for PP2A in the maintenance of active zones. The receptor phenotype is modified by genetic interactions with APC2, a homolog of the multifunctional adenomatous polyposis coli protein (APC), and glycogen synthase kinase-3beta (GSK-3beta), both members of a signaling cassette implicated in neuronal polarity and axonal outgrowth. Heterozygous mutants in APC2 enhance the receptor phenotype, whereas inhibition of GSK-3beta suppresses the receptor phenotype. These results suggest a novel function for APC/GSK-3beta signaling. They are consistent with a model in which GSK-3beta and PP2A co-regulate phosphorylation dynamics of substrate(s) required for normal active zone development, while APC acts to promote PP2A function. As a regulator of synaptic development, PP2A is thus required for normal synaptic structure, both at the level of the synaptic terminal as well as active zones and receptors. |
