Beug, Shawn Terrence2013-11-082013-11-0820092009Source: Dissertation Abstracts International, Volume: 71-06, Section: B, page: 3654.http://hdl.handle.net/10393/29897http://dx.doi.org/10.20381/ruor-19965The Sonic hedgehog (Shh) morphogen plays an important role in shaping central nervous system (CNS) development. While the function of Shh signalling in many tissues is well understood, the intracellular regulation of Shh trafficking during these processes is relatively unknown. To examine the mechanisms of Shh trafficking in neurons, I generated cDNA constructs coding for Shh isoforms, transfected these into differentiated PC6 cells and primary hippocampal neurons, and monitored protein distribution by fluorescence microscopy and biochemistry. Shh is distributed along axons, dendrites and at the neurite terminals in neurons and is associated intra- and extracellularly with lipid raft markers. Intracellularly, wild type Shh associates with synaptic-like microvesicles and dense core granules (DCGs), and is transported with both fast and slow kinetics. Inhibiting Shh posttranslational modification, including cleavage and lipidation, resulted in the accumulation of Shh morphants with DCGs and lack of cell surface accumulation. Consistent with the enrichment of Shh morphants in DCGs, secretion of Shh could be inducted under depolarizing conditions. Taken together, these observations suggest that long-range Shh transport and signalling in neurons involves trafficking to the regulated secretory pathway and cell surface accumulation of Shh on axons, and suggests a link between neuronal activity and Shh secretion. The second aim involved a GST-pull down affininity purification method to identify potential Shh interacting proteins that could mediate Shh intraneuronal transport. Recombinant Shh-N or Shh-C fused to GST were used as bait and incubated with solubilised membrane fractions from the perinatal rat brain. Interacting proteins were resolved by SDS-PAGE, and individual bands were excised and identified by mass spectrometry. From this screen, 22 proteins were identified to be potential Shh interacting factors. Several candidates specific for Shh-N or Shh-C were identified, and I selected three candidates, Sortilin (Sort1), Glypican 5 (Gpc5) and Low density lipoprotein receptor-related protein 1 (Lrp1), for validation by coimmunoprecipitation. These candidates demonstrate potential to be direct Shh interacting partners that are involved in Shh trafficking, extracellular transport and reception within CSN development. Overall, these results indicate that Shh transport and release is tightly regulated and may be differentially secreted from the cell body or neurite, and rely on context- and tissue-specific protein:protein interactions for appropriate vesicular targeting, intraneuronal trafficking, extracellular transport and reception.211 p.enChemistry, Biochemistry.Thesis