Regulation of protein trafficking and dendrite branching by postsynaptic proteins

The establishment of neuronal connections during the development of the mammalian brain influences adult brain function. After development, synapses are subjected to continuous modification and maintenance throughout adulthood. Thus, homeostasis at the synaptic level requires trafficking of receptors, signaling and scaffolding proteins to and from the postsynaptic specialization. Therefore, the focus of my three research projects has been to investigate how proteins are trafficked to the synapse and how connections are made while the synapse develops.; My work first focused on the regulation of the trafficking of N-methyl-D-aspartate receptors (NMDARs), a subtype of glutamate channels. NMDARs have been implicated in learning, memory, Alzheimer's Disease and schizophrenia. We identified snapin (SNAP-25 interacting protein) as a binding partner to the NMDAR subunit, NR1. This interaction takes place through the coiled-coil domain of snapin and the first intracellular loop and C0-C1-C2 tail of NR1. Overexpression of snapin in cultured hippocampal neurons causes an activity-independent decrease in NR1 clusters per area dendrite. These NR1 clusters have increased cell surface expression and show a higher degree of apposition to synaptophysin immunostaining compared to NR1 clusters in neurons expressiog GFP.; In addition, I also studied the regulation of the interaction between PSD-95 and the exocyst subunit, sec8, by the PSD-95 interacting protein cypin. PSD-95 is a member of the membrane-associated guanylate kinase (MAGUK) family. MAGUK proteins function as scaffolds that anchor signaling proteins in close proximity to each other to mediate downstream signaling. We found that sec8 and PSD-95 not only interact in the brain but also co-localize at the microtubule organization center (MTOC), cell body, and neurites of PC12 cells. The interaction between sec8 and PSD-95 is regulated by cypin binding to PSD-95.; Lastly, I studied the regulation of dendrite branching by snapin. In this study, we found that the carboxyl half of snapin binds to the CRMP domain of cypin, which is responsible for tubulin heterodimer binding to cypin. As a result, snapin decreases cypin-promoted microtubule assembly in a cell-free assay. In cultured hippocampal neurons, overexpression of snapin results in a decrease in the number of primary dendrites and an increase in the probability that the primary dendrites will branch.