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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31NS068038-01
Application #
7811357
Study Section
Special Emphasis Panel (ZRG1-ETTN-G (29))
Program Officer
Riddle, Robert D
Project Start
2010-02-01
Project End
2013-01-31
Budget Start
2010-02-01
Budget End
2011-01-31
Support Year
1
Fiscal Year
2010
Total Cost
$27,928
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Otolaryngology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Kokel, David; Cheung, Chung Yan J; Mills, Robert et al. (2013) Photochemical activation of TRPA1 channels in neurons and animals. Nat Chem Biol 9:257-63
Coutinho-Budd, Jaeda C; Snider, Samuel B; Fitzpatrick, Brendan J et al. (2013) Biological constraints limit the use of rapamycin-inducible FKBP12-Inp54p for depleting PIP2 in dorsal root ganglia neurons. J Negat Results Biomed 12:13
Coutinho-Budd, Jaeda; Ghukasyan, Vladimir; Zylka, Mark J et al. (2012) The F-BAR domains from srGAP1, srGAP2 and srGAP3 regulate membrane deformation differently. J Cell Sci 125:3390-401
Charrier, Cecile; Joshi, Kaumudi; Coutinho-Budd, Jaeda et al. (2012) Inhibition of SRGAP2 function by its human-specific paralogs induces neoteny during spine maturation. Cell 149:923-35