Oregon Health & Science University
Neuroscience Graduate Program
School of Medicine
During formation of the nervous system in many organisms, neurons must migrate over long distances to reach their appropriate locations and establish proper connections with their synaptic targets. To facilitate their guidance in the developing embryo, neurons express a variety of receptors that can both sense extracellular cues and elicit intracellular signaling cascades that mediate stage-specific types of motile behavior, including cellular migration, axonal outgrowth, and synaptic formation. Amyloid precursor protein (APP) has been shown to regulate numerous aspects of neuronal motility in vitro, supporting its role as a guidance receptor in the developing nervous system. However, elucidating the function of APP in vivo has been complicated by the expression of two closely related genes (APLP1 and APLP2) in mammals that share partially overlapping functions with APP, and the discovery that different APP isoforms are expressed by multiple cell types besides neurons in vertebrate model systems. As an alternative strategy, I have utilized the developing enteric nervous system (ENS) of the hawkmoth, Manduca sexta. During formation of the ENS, a population of approximately 300 neurons (EP cells) reaches their correct target locations on the gut musculature by migrating along an identified set of muscle band pathways that are permissive to their growth. Previous studies in the Copenhaver laboratory demonstrated that APPL (APP-like), the sole ortholog of APP in insects, is expressed by the EP cells during active phases of migration and outgrowth, co-incident with their expression of the heterotrimeric G protein Goα. Although activation of Goα has been shown to induce stalling responses in migrating EP cells and their axons, the upstream receptor in this pathway has remained unidentified. Work from several groups has suggested that APP functions as an unconventional G protein-coupled receptor, capable of regulating a variety of cellular responses via the eterotrimeric G protein, Goα. However, most of these investigations employed artificial liposome preparations and transfected cell lines, leaving the validity of this model in doubt. To test whether insect APPL might also function by regulating Goα-dependent aspects of neuronal migration and outgrowth, I discovered that blocking APPL signaling in the EP cells induced a pattern of ectopic, inappropriate growth and migration in the Manduca ENS, identical to the effects of inhibiting Goα activity. Using a combination of biochemical, pharmacological, and bimolecular luorescence complementation assays, I showed that APPL directly interacts with Goα in the developing nervous system, in cell culture, and at synaptic terminals within the fly brain, and that this interaction is regulated by Goα activity. Corresponding assays using both murine and human brain tissue samples revealed that Goα (but not other G proteins) also interacts with endogenously expressed APP, and that this interaction is specific to the full-length (transmembrane) protein. These results support the model that APP family proteins function as Goα-coupled receptors that prevent neuronal migration and outgrowth from occurring in nonpermissive regions. To date, authentic ligands for APP family proteins are not known. As an alternative approach for activating APPL, I used established methods for antibody crosslinking to induce APP signaling in murine neuronal cultures. These studies revealed a role for APP in promoting Go-dependent growth cone collapse and retraction. To test whether similar responses could be elicited in vivo, I treated cultured Manduca embryos with Fc fusion constructs of one of its candidate ligands, Mscontactin. Treating premigratory EP cells with Mscontactin-Fc resulted in a stalling response, consistent with the expected effect of activating APPL in this system. Whether APPL-Goα signaling lies downstream of Mscontactin in mediating this effect is still to be determined. Lastly, using a combination of biochemical and mmunohistochemical methods, I identified APP phosphorylation at one specific residue within its ytoplasmic domain (Thr668) as a potential regulator of APP-Goα nteractions. Unexpectedly, I discovered that Goα preferentially interacts with the nonphosphorylated form of Thr668, suggesting that kinase-dependent phosphorylation of APP at this site may provide another mechanism for regulating APP-Goα signaling. In combination, this work identifies a role for the evolutionarily conserved APP family in regulating key aspects of neuronal motility and outgrowth by functioning as Goα-coupled receptors. Future studies will be needed to determine how APP-Goα signaling is regulated in the developing nervous system, and how perturbations in this signaling pathway may contribute to the progression of neurodegenerative diseases in the adult brain (including Alzheimer’s disease).