Jaeda Coutinho-Budd, Ph.D. - US grants

DESCRIPTION (provided by applicant): Formation of functional neuronal circuits in the cerebral cortex involves coordinated migration of pyramidal neurons to their final location, as well as subsequent projection of neurites to their targets. Defects in both neuronal migration and morphogenesis can lead to developmental neuropathologies such as autism, lissencephaly, mental retardation, and schizophrenia. By elucidating the mechanisms that regulate these processes, we can improve our understanding of the developmental mechanisms underlying some of these socially-devastating diseases. Towards this aim, I will examine the role and regulation of the protein, slit- robo GTPase Activating Protein 2 (srGAP2), during neuronal differentiation. Recently, the Polleux lab has uncovered the function of srGAP2 as a negative regulator of cortical neuron migration and a positive regulator of neurite initiation and branching in the same neurons. Moreover, we found that srGAP2 increases neurite initiation and branching in cortical neurons through its ability to form actin-rich, filopodia- like membrane protrusions. Our preliminary results show that this function is primarily carried out by its N- terminal, membrane-deforming F-BAR domain. Interestingly, a mutation preventing its Src Homology 3 (SH3) domain to interact with its binding partners blocks the function of srGAP2 in neurite branching and neuronal migration. Conversely, a truncated form of srGAP2 lacking the C-terminal end, including the SH3 domain, increases neurite branching and blocks neuronal migration as potently as full-length srGAP2. In order to explain these results, we hypothesize that the C-terminal domain of srGAP2 interacts with, and inhibits, the protruding activity of its F-BAR domain. In this 'auto-inhibitory'model, binding of specific interactors to the SH3 domain activates srGAP2 by releasing the F-BAR domain, allowing it to dimerize and deform membrane. To test this hypothesis, I will transfect mutant forms of srGAP2 into cortical neurons in order to perform a structure/function analysis aimed at identifying the residues involved in the auto-inhibition and activation of srGAP2 (Aim 1). Additionally, I will use this technique, along with co-immunoprecipitation, to identify neuron-specific, SH3-interacting proteins and determine if these candidate proteins are involved in activating srGAP2 during cortical neuron morphogenesis (Aim 2). Finally, we have acquired a targeted srGAP2 knockout mouse to help establish the role of srGAP2 in vivo (Aim 3). Enhancing our understanding of the mechanisms underlying srGAP2 function in neurite formation and neuronal migration will improve our understanding of the molecular mechanisms underlying cortical development.

DESCRIPTION (provided by applicant): Local regional spread of tumors is driven by increased tumor cell invasiveness, as is metastasis, whereby primary tumors spread to secondary tissues. Precisely how tumor cells become invasive is poorly understood, but understanding this transformation remains a major goal is basic biomedical research. The term glioma broadly describes a category of molecularly heterogeneous tumors, typically arising from glial cells such as astrocytes or oligodendrocytes. Glioma can be further broken down into four graded classifications (I-IV), which correspond increasingly with malignancy, culminating in glioblastoma (grade IV)1. Not only are many high-grade gliomas typically resistant to chemotherapy, but their invasive nature makes surgical removal nearly impossible, leading to poor patient prognosis2. Prior to tumorigenesis, glia play important roles in regulating nervous system function, including: providing trophic factors for neurite growth and guidance4-6, and facilitating synapse formation, maturation, and plasticity9. However, genetic lesions transform these beneficial cells into destructive cancers through a variety of unidentified mechanisms. It is therefore paramount to better understand the basic molecular and genetic mechanisms that regulate glial proliferation, growth, and infiltration in order to determine how these processes go awry in glial disease. While multiple model systems are available to study glia in health and disease, this work uses Drosophila melanogaster because it allows for genetic manipulation on a genome- wide scale in vivo. Additionally, Drosophila offers an unparalleled array of powerful molecular-genetic tools with which to dissect cellular mechanisms and gene function in vivo. Cortex glia are a striking subclass of glia that extend fine processes to form a lace-like structur that infiltrate the cortex and encapsulate neuronal somas10,11. While cortex glia are a relatively understudied glial cell type of the Drosophila nervous system, they are a good model cell to investigate mechanisms of glioma because they are comparable to subsets of mammalian glia that give rise to glioma14, they have the ability to self-proliferate15, and have protrusions that infiltrate between other cells analogously to invadopodia of metastasizing tumor cells. This proposal aims to define the cellular, genetic, and molecular mechanisms involved in glial development, growth, and infiltration. Aim 1 will define the developmental patterns of cortex glial growth and infiltration. Aim 2 will determine how loss of ?-SNAP impacts cortex glial growth, and surrounding neurons. Finally, Aim 3 will determine the mechanism(s) by which ?-SNAP functions to regulate glial growth and infiltration. Such investigations are vital to enhance our understanding of the mechanisms of glial growth, and potential to advance the identification of biomarkers capable of earlier glioma detection, and aid in discovering new and effective therapeutic targets for suppression of glial cell invasive behavior in glioma.

PROJECT SUMMARY/ABSTRACT All major mammalian glial subtypes of the central nervous system (CNS) make direct contacts with neuronal cell bodies; however, how glia support and communicate with neuronal somas is vastly understudied compared to glial interactions at synapses or axons. The overarching goal of this project is to gain a deep mechanistic understanding of glial development, communication, and function at neuronal cell bodies to begin to fill in the gaps of how these associations regulate CNS health and dysfunction. Drosophila glia demonstrate remarkable similarity to a number of mammalian glial subtypes measured by morphological, functional, and molecular criteria. Among these is cortex glia, a glial subclass that forms a lace-like meshwork to individually ensheath nearly every neuronal cell body in the CNS. We recently developed new genetic tools to manipulate gene function with remarkable specificity in Drosophila cortex glia, and now have a powerful system in which to study glial cell development and neuron-glia interactions at neuronal cell bodies in vivo. In addition to regulating neuronal health and behavior, cortex glia provide metabolic support to neurons, regulate neuronal ion and nutrient balance, engulf neuronal debris, and can therefore inform the interrogation of multiple vertebrate glial cells that interact with neuronal cell bodies. We previously demonstrated that when cortex glia lack a single secreted neurotrophin, Spätzle 3 (Spz3), they take on a globular appearance and no longer wrap neuronal cell bodies. The loss of Spz3 and these glial-somal interactions leads to widespread nervous system dysfunction, including increased neuronal cell death, locomotor impairment, and aberrant growth of surrounding healthy glial cells. We propose to use powerful in vivo genetic tools available in Drosophila, along with a variety of techniques in cellular and molecular biology, biochemistry, and imaging to elucidate the mechanisms of glial-somal interactions that maintain neuronal health in a live, intact nervous system. Specifically, we will dissect the mechanisms that regulate the maturation and distribution of this neurotrophin to maintain glial contact at neuronal cell bodies (Aim 1), define how this neurotrophin signals to its receptor to support glial morphology, somal interactions, and neuronal health (Aim 2), and finally, we will determine how nearby glial cells compensate when glial-somal signaling and associations are impaired (Aim 3). These findings will begin to shed light on an understudied, yet important phenomenon by providing a foundation for elucidating the cellular and molecular underpinnings of glial interactions with neuronal cell bodies in the healthy and diseased CNS.