UC San Diego

Astrocytes secrete proteins that are critical for normal neuronal development, but while work has identified some important proteins in this process, little is known about the protein secretion of astrocytes at time points when they are most actively involved in supporting neuronal outgrowth and synapse formation. This dissertation seeks to elucidate the protein secretion profile of murine astrocytes at postnatal day 7, using a newly developed immunopanning technique to prospectively isolate astrocytes. These astrocytes are maintained in vitro in minimal media in order to examine their secreted proteins to identify key mediators of neuronal development. By comparing the protein secretion profiles of wild-type astrocytes to those of astrocytes isolated from mouse models of genetic neurodevelopmental disorders associated with autism, this work also seeks to identify alterations in protein secretion in disordered astrocytes that play a role in the pathology of autism.

Using mass spectrometry, I identified over 1200 proteins secreted by astrocytes in vitro and nearly 100 proteins that show differential secretion patterns in wild-type astrocytes compared to three different genetic neurodevelopmental disorders (Rett Syndrome, Fragile X Syndrome, and Down Syndrome). Furthermore, I identified two proteins that show dramatically increased secretion in astrocytes from all three disorders compared to wild-type: insulin-like growth factor binding protein 2 (IGFBP2) and bone morphogenetic protein 6 (BMP6), implicated in the pathology of Rett Syndrome and Fragile X Syndrome, respectively. Wild-type astrocyte conditioned media (ACM) is capable of supporting neuronal outgrowth compared to a minimal media while disordered ACM results in outgrowth deficits. Addition of IGFBP2 to wild-type ACM leads to outgrowth deficits in vitro, which can be blocked with the application of an IGFBP2- neutralizing antibody. Addition of IGFBP2-neutralizing antibody can rescue deficits induced by Rett Syndrome but not Fragile X Syndrome ACM. Conversely, BMP6 treatment of wild-type astrocytes generates ACM that is similarly incapable of supporting neuronal outgrowth, and blocking BMP activity in Fragile X astrocytes with the application of noggin protein can rescue the aforementioned outgrowth deficits. Thus I have identified astrocyte-secreted proteins that may prove to be critical for neuronal development, and provide targets for therapeutic treatments of these genetic neurodevelopmental disorders.