Dissecting cellular mechanisms of neurite formation in the developing cortex

Neurite formation is an early and critical step in neuronal polarization, upon which subsequent stages rely. We identified a role pigment epithelium-derived factor (PEDF) in regulating neuromorphogenesis. PEDF is a secreted glycoprotein that is encoded in the clinically relevant Miller-Dieker syndrome (MDS) critical region of chromosome 17p13.3, which is deleted or duplicated in a variety of neurodevelopmental disorders. We found that two of the four known PEDF receptors, ribosomal protein SA (Rpsa) and plexin domain-containing protein 1 (Plxdc1), have roles in neuromorphogenesis. Rpsa regulates neurite initiation and elongation, as well as arborization and spine formation via PEDF. Rpsa knockdown causes defective sub-threshold spontaneous calcium signaling. Additionally, we show that integrin [alpha]6 (Itga6), which is known to interact with Rpsa on the plasma membrane, stabilizes Rpsa on the membrane where it must be localized to bind PEDF. Rpsa and Plxdc1 signaling are distinct and regulate specific aspects of development. Plxdc1 overexpression has the unique ability to drive neurite formation. This distinctive phenotype allowed for the effect of a variety of kinase inhibitors to be assessed, leading to the identification of Akt as a kinase specifically involved in Plxdc1 overexpression-driven neurite formation. Furthermore, proteomic analysis of the cytoskeletal fraction revealed a 3.43 fold increase in activity-dependent neuroprotector homeobox (Adnp) following Plxdc1 overexpression, identifying Adnp as a putative component of the Plxdc1 signaling pathway that can directly bind microtubules, facilitating neurite formation. To identify additional kinase inhibitors that may be useful therapeutic targets, the control for the above mentioned kinase inhibitor screening was also used to identify kinases involved in neurite formation induced by FBS-depletion, which is a widely used method for inducing N-2a cell differentiation. We identified the Aurora and Nuak kinases as candidates for further investigation. Using primary cortical neurons, we found that Aurora A has a role in regulating neurite initiation, while Aurora B and C have roles in regulating neurite initiation and elongation and all Aurora kinases regulate arborization. Additionally, Nuak 1 and 2 regulate neurite initiation and elongation and arborization. Together, this work has identified several important molecules that function in regulating unique aspects of neurite formation. We have elucidated the role of PEDF signaling via its membrane receptors Rpsa and Plxdc1 during cortical neuronal development, presenting possible insights into the etiology of neurodevelopmental disorders such as MDS. Additionally, we identify multiple kinases which may be useful therapeutics in treating conditions where aberrant axonal sprouting or failed regeneration of injured axons occur, such as traumatic injury, neurodegenerative disease, and seizures.