Title: Molecular mechanisms underlying synapse development
Description: Mammalian nervous system development occurs through an intricate genetic program that ensures that brain structures and cells form and are in the appropriate place by the time of the birth of the organism. The initial steps in the formation of the nervous system are the induction and patterning of neurogenic regions and the generation of neural progenitors which give rise to neurons and glia. These early steps are followed by periods of neuronal migration, axon guidance, and synaptogenesis. Following initial development, postnatal sensory, cognitive, and motor experiences play a key role in shaping neuronal circuitry during the early stages of nervous system development, and later in life, sensory experiences lead to the formation of long-lasting memories and alterations in the behavior of adult organisms. To begin to address this question we investigated the signaling mechanisms by which the Eph family of receptor tyrosine kinases mediates axon guidance.

Yeast two-hybrid screening identified the Rho family GEF ephexin1 as an EphA4-interacting protein. In the first part of this thesis, we demonstrate that ephexin1 is a critical regulator of Eph-receptor mediated axon guidance. In the absence of ephrin contact, ephexin1 promotes growth cone extension by activating the Cdc42 and Rac1 GTPases. Ephrin engagement of Eph receptors on the growing axonal growth cone promotes the phosphorylation of ephexin1 on Tyrosine-87 which preferentially activates ephexin1 exchange towards RhoA, but not towards Rac1 and Cdc42. This switch in RhoGTPase family activation induces growth cone collapse and repulsion. The importance of ephexin1 for ephrin-mediated axon guidance was demonstrated in vivo, as ephexin1-deficient mouse retinal ganglion cells are unable to respond to ephrinA guidance signals, and in the chick, knockdown of ephexin leads to motor neurons aberrantly projecting their axons into the limb mesoderm. Taken together, our results demonstrate a critical role for the Rho family GEF, ephexin1 in Eph-receptor mediated axon guidance and begin to elucidate the molecular mechanism by which axons are ultimately guided to the appropriate location within the nervous system. Once the axon reaches its final destination, the processes of synapse formation, maturation, and refinement begin. Throughout development, neuronal activity modulates both the number and strength of synaptic connections. This process is extremely complex and involves many different types of molecular modifications including receptor trafficking, local translation, protein turnover and new gene synthesis. An earlier study in our laboratory revealed that the activity-regulated transcription factor, Mef2 is a key mediator of activity-dependent synapse development.

In response to neurotransmitter release, Mef2 initiates a program of gene transcription that restricts the number of synapses formed by a neuron. One of the components of this program is Ube3A and in the second half of my thesis I have investigated the role of the E3 ubiquitin ligase, Ube3A in synapse development and function. Mutation of Ube3A in humans results in the neurodevelopmental disorder Angelman Syndrome which is characterized by severe mental retardation, ataxia, hyperactivity, and frequent seizures. At the time that we initiated these studies although it was known that mutation of Ube3A resulted in Angelman Syndrome, very little was known about the function of Ube3A during nervous system development or why mutation of Ube3A results in the cognitive impairment observed in individuals with Angelman Syndrome. In the present study, we have demonstrated that the expression of Ube3A is induced by experience-driven neuronal activity, and have shown that Ube3A is a critical regulator of excitatory synapse development. Ube3A deficient neurons have significantly more excitatory synapses than their wild type counterparts and also express significantly fewer AMPA receptors on their cell surface. The ability of Ube3A to modulate synapse number and AMPA receptor surface expression is dependent upon Ube3A's ubiquitin ligase activity and we have identified Arc as a substrate of Ube3A that mediates its effect on synapse development.

In the absence of Ube3A, higher levels of Arc, a key mediator of AMPA receptor endocytosis, accumulate in response to experience-dependent neuronal activity and consequently there is a decrease in AMPA receptor at synapses. This reduction in synaptic AMPA receptor expression may trigger a homeostatic response that results in an increase in the number of synapses that are formed or maintained by the neuron. Taken together these findings suggest that Angelman Syndrome may be caused by the disruption of a crucial step in experience-dependent synaptic development and uncover an important role for Ube3A in synaptic function.