Elke Stein, Ph.D. - US grants

In the developing nervous system, axons grow long distances over complex terrains, making use of intermediate targets to simplify their navigation into short, controllable segments. One of these intermediate targets is the ventral midline of the nervous system. Axonal growth cones that cross the midline change their responsiveness to secreted midline guidance cues: They become repelled by Slit and simultaneously lose responsiveness to the attractant netrin-1, which initially guided them to the midline. These mutually reinforcing changes then help to expel growth cones from the midline by making a once-attractive environment appear repulsive. Initial studies revealed that these two changes are interlocked, and thus activation of the Slit receptor Robo1 can silence the attractive effect of netrin-1, but not its growth-stimulatory effect, through direct binding of the intracellular domain of Robo1 to that of the netrin receptor DCC. Interestingly, recent genetic evidence supports a crucial role for Robo1 in midline guidance and implies that Robo1-mediated repulsion is essential for expelling growth cones from the midline. Dr. Stein hypothesizes that the Slit receptor Robo1 participates in silencing netrin-mediated attraction, as well as in expelling growth cones from the vertebrate midline, that the Robo1 cytoplasmic domain is organized in modules and that distinct modules are essential for signaling Robo1-dependent silencing and repulsion. Dr. Stein further proposes that silencing and repulsion are signaled through distinct, but overlapping, pathways.

Dr. Stein plans to use biochemical and functional approaches to learn how Robo1 signals repulsion. The Stein laboratory will first elucidate the receptor mechanism that Robo1 utilizes to signal repulsion and will further investigate the functional contribution of known and novel, recently identified partners associating with the intracellular domain of Robo1 to signaling Robo1 repulsion in vertebrates, using biochemical and functional assays. The results of this study will give insights into which intracellular signaling pathways the Slit receptor Robo1 couples to signal repulsion and will be an entry point to understand the molecular basis of silencing. The work will help to consolidate the general understanding of the intracellular signaling pathways to which axonal guidance receptors, such as Robo1, couple to signal repulsion and may elucidate how this information is translated into directed guidance at the midline.

Dr. Stein is training undergraduates, graduate students, postdoctoral fellows and research assistant in her laboratory, including members of underrepresented minority groups. The project will support training of students in areas of biochemistry, cellular and developmental neurobiology, preparing them for advanced scientific careers. Dr. Stein will also integrate education with her research program by developing up-to-date instructional material for an advanced course in neuronal cell biology and developmental neurobiology, a research seminar for undergraduates and a University-wide special-topic seminar discussing breakthrough technologies in modern developmental biology, as well as initiating a community outreach program for brain awareness. Finally, fundamental knowledge about the molecular and cellular mechanisms governing nervous-system wiring provides a firm foundation for developing future neurobiological applications of general benefit to society.

[unreadable] DESCRIPTION (provided by applicant): In the developing nervous system, nerve cells and axons respond to a variety of attractive and repulsive guidance cues en route to their final destination. On their journey they follow a complicated trajectory using in some circumstances multiple cues, in others switching their responsiveness to a particular bifunctional cue, acting over short and long range. An essential step in obtaining a better understanding of the molecular mechanisms that underlie these events is to identify receptor complexes that couple guidance cues to directed guidance. Uncovering the molecular mechanisms that translate an extracellular cue into a change in the growth cone cytoskeleton is likely to lead to a better understanding of certain developmental disorders, and also to contribute towards development of therapeutic interventions to improve nerve regeneration following injury. The central aim of this proposal is to characterize receptor complexes that mediate responses of growth cones to the bifunctional guidance cue Netrin-1 in the developing nervous system.Netrin-1 attracts several classes of axons, while repelling others. Netrin receptors of the DCC family are implicated in mediating both attraction and repulsion; in contrast members of the UNC-5 family are implicated in signaling repulsion alone or in combination with DCC. How UNC5 signals repulsion independent of DCC is not known. Here we hypothezise that UNC5 signals two types of DCC-independent repulsion, through homomultimerization of UNC5A and through heteromultimerization of UNC5B-D with the orphan receptor PUNC. We are determined to elucidate how Netrin-1 mediates axonal repulsion. We will (I) characterize DCC- independent repulsion in vertebrates spinal neurites in vitro, (II) define the receptor complexes mediating DCC independent repulsion by using a combination of biochemical and genetic approaches and (III) evaluate the function of these receptors in Netrin-1 signaling in vitro and in vivo by utilizing directional and outgrowth responses of primary neuronal cultures in combination with loss- and gain-of-function approaches. This work will provide the foundation for understanding how neurons translate a bidirectional cue, such as Netrin-1, into directed guidance. In addition, it should be of more general importance in providing insights into the underlying receptor mechanism used by bifunctional cues to guide axons during neurodevelopment. [unreadable] [unreadable]