| Summary: | 碩士 === 國立臺灣大學 === 分子醫學研究所 === 99 === In response to environmental and physiological stimulations or fluctuations, the synapse has to perform plasticity and also maintains homeostasis. Accumulating evidences support the role of the ubiquitin-proteasome system (UPS) in regulating synapse formation and remodeling, thus maintaining long-term neural circuit plasticity and homeostasis. Here we characterize the function of BTB-Kelch protein, the substrate receptor in the Cullin3 (Cul3)-organized ubiquitin E3 ligase, in the formation and neurotransmission of Drosophila neuromuscular junctions (NMJs). Mutant NMJs lacking henji activity show a dramatic increase in bouton number, including the appearance of numerous satellite boutons. Ultrastructurally, the electron-dense membrane area delineating the presynaptic active zone and the postsynaptic density (PSD) is expanded and the periactive zone is concurrently decreased in henji mutants. The PSD houses glutamate receptors (GluRs) IIA and IIB that show distinct transmissions at Drosophila NMJs. In henji mutants, the GluRIIA abundance is upregulated and the GluRIIB is downregulated. Our electrophysiological results also support a composition shift toward a higher GluRIIA/IIB ratio at henji mutant NMJs. By rescue experiments, we show that Henji acts in the postsynapse to regulate proper NMJ growth and GluRIIA/IIB composition.
We further show that Henji controls NMJ growth and GluRIIA/IIB ratio by downregulation of dPak at PSDs. The postsynaptic dPAK marks the PSD area and regulates the GluRIIA abundance. Losing one copy of dpak suppressed the bouton phenotype and GluRIIA abundance in the henji mutant. Also, the intensity and area of dPAK punctates at PSDs were increased in henji mutants. We found that dPAK interacts with Henji, which promotes ubiquitination and degradation of dPak. Therefore, Henji acts at PSDs to restrict both the presynaptic bouton growth and the postsynaptic GluRIIA clustering via controlling dPAK protein level.
Several questions remain to be addressed: the subcellular localization of Henji; how dPAK is regulated by Henji during its activation; and how the GluRIIA/IIB balance is controlled by Henji-regulated dPak levels. Some preliminary results and future works will be presented in the thesis.
In addition, we also addressed the role of integrin betanu in restricting NMJ growth. In the loss of betanu activity, drastic increase in bouton number was observed. Further, we dissected the downstream signaling of betanu/FAK56 and reported a bifurcating cascade of NF1-regulated cAMP/PKA and Vap-mediated Ras/MAPK pathways.
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