Paul C. Letourneau - US grants

DESCRIPTION (provided by applicant): A growth cone is the motile tip of a growing axon or dendrite. Proper assembly of neural circuits depends on navigation by growth cones during development. Five behaviors of growth cones are critical in forming neural circuits: migration, turning, branching, retraction, and transformation to synaptogenesis. A growth cone detects environmental cues and responds by regulating these five behaviors. In addition to improving our understanding of how the brain develops, we believe elucidating the mechanisms of growth cone guidance will help in designing strategies to promote repair and regeneration of damaged nervous tissue. We will pursue two aims to test our main hypothesis that neurotrophins modulate the responses of growth cones to other guidance cues via cytoplasmic signaling pathways that regulate the dynamics of cytoskeletal components: (1) Elucidate the role of neurotrophins in regulating growth cone responses to multiple guidance cues. We will test the hypothesis that the p75 neurotrophin receptor is involved in neurotrophin regulation of growth cone behavior. We will test the hypothesis that neurotrophin regulation of growth cone behaviors involves binding to p75, activation of protein kinase A (PKA) and inhibition of p75-mediated activation of RhoA GTPase. (2) Elucidate the cytoskeletal changes and interactions that accompany growth cone collapse and retraction. We will test the hypothesis that myosin II contractility drives growth cone collapse, retraction and cytoskeletal rearrangements. We will test the hypothesis that neurotrophins stabilize actin filaments against collapse by preventing loss of actin monomer from filament pointed ends. These studies will involve tissue culture of sensory, retinal and ciliary neurons from chick embryos and sensory and retinal neurons from mice with mutant p75 and trkB genes. Explants of neural tissues will be prepared and growth cone behaviors will be analyzed by video microscopy and quantitative fluorescence microscopy. Responses of growth cones to several molecular guidance cues will be determined, and the activities of PKA and Rho family GTPases will be measured. Cytoskeletal organization will be analyzed in growth cones treated with neurotrophins and with negative guidance cues.

An International Conference on Nerve Growth Cones is being planned for the Cajal Institute, Madrid, SPAIN from October, 1990. The goal of the Conference is to bring together top researchers on nerve growth cones to synthesize current knowledge, develop new concepts of growth cone function, and crystallize strategies for future experimentation. This meeting is especially timely, because techniques and tools now coming available from chemistry, molecular biology, physiology, optics and computerized imaging technology are allowing significant gains in elucidation of how growth cones mediate the intricate construction of nervous systems. A meeting of scientists who use these diverse methods will generate new insights and collaborations. The conference will cover the following topics: internal chemistry and physiology of growth cone activity, external control of growth cone behavior, growth cone activity in developing organisms as neural circuits are constructed, and growth cone activity in adult organisms and after neural circuits are damaged. Thus, this meeting will help explain how the human brain grows, adapts, learns, and perhaps can be repaired. Twenty five speakers have been invited and have greed to speak at the meeting and also write a chapter for a published monograph. In addition, poster exhibits will be presented. Neurobiological interest in growth cone activity has grown tremendously, and this first meeting devoted solely to growth cones will receive wide notice and will enhance research activity in the area for several years.

The differentiation of neurons, the acquisition of specific neuronal shapes and the formation of synapses are regulated by interactions of neurons with other cells and components of extracellular matrices. Our preliminary results implicate a cell surface heparan sulfate proteoglycan (HSPG) in FN-mediated neuronal cell adhesion and neurite outgrowth. In these studies, we have identified a synthetic heparin binding Peptide from Fibronectin (FN) which promotes the adhesion of a neuronal cell line, B104, and promotes neurite outgrowth from dorsal root ganglion and spinal cord neurons from chick embryos. We propose to use the neuroblastoma cells and primary neurons to further define the molecular bases for proteoglycanmediated neuronal cell adhesion to the 33 kD heparin binding fragment of FN. Specifically, we propose: 1 . To use overlapping synthetic peptides to further define the heparin/proteoglycan binding and cell adhesion promoting activity of FN-C/H 11. 2. To isolate cell surface HSPG from B 1 04 neuroblastoma cells and to generate polyclonal and monoclonal antibodies against this HSPG. 3. To use anti-HSPG polyclonal and monoclonal antibodies to partially characterize HSPG expressed by neuroblastoma cell lines. 4. To use anti-core protein antibodies to determine the localization and functions of core protein on neurons in fetal and neonatal nervous system. These studies will include examination of the in vitro distribution of HSPG on specific cell types and on cell bodies, neurites, growth cones, determination of spatial and temporal correlations in vivo, and experiments to assess the function of the HSPG in neuronal interactions with fibronectin.

9805835 Hu The long-term objective of this project is to develop a method of directing nerve axonal growth within three-dimensional matrices for tissue engineering applications. To facilitate the selective deposition of biologically active molecules in a three-dimensional structure, a photochemical technique will be employed that relies on a penetrating laser to spatially control the binding of bioactive molecules. This method can be applied to deposit virtually any molecule that can be conjugated to a photolinker. The immediate objective of this project is to apply the developed method to investigate the factors critical to path finding in both two-dimensional surfaces and three-dimensional structures. ***

This proposal was received in response to the announcement NSF 01-157.

They have organized a Nanoscale Interdisciplinary Research Team (NIRT) motivated by the hypothesis that understanding of the roles that concentration gradients of signal molecules play in cell dynamics, neuronal axon growth in particular, with unprecedented spatial resolution and
chemical control is very important. The team consists of four PIs from Chemistry, Neuroscience, and Mechanical Engineering, with expertise in surface functionalization and biomolecule immobilization, neuronal axon growth mechanisms, and nano-delivery and characterization tools for biomolecules. The combination of expertise and synergy from this collaboration will allow them to develop methodology for the generation of chemical and biochemical gradients that possess the following attributes: (a) the gradients are supported on a solid surface for precise control and quantification; (b) the gradients possess cellular (~um) and sub-cellular (~102 nm) spatial resolution; (c) arbitrary or "designer" gradient profiles, including digital (discrete) or analog (continuous) gradients, are possible; (d) the method are not molecule-specific and the gradients may include multiple components.
The proposed method will be based on a precision nano-delivery tool, an electrospray system capable of 10-18 liter volume resolution, to deliver chemical or biological samples onto a functionalized solid surface. A "designer" gradient will be generated by a programmed voltage
pulse-train in combination with programmed movement of the sample. Monolayer assemblies on
the solid surface will be designed to confine the nano-droplet for high resolution, to immobilize
signal molecules in the nano-droplet, and to provide chemical or biological compatibility for cell
adhesion and growth. They plan to use neurons as a model biological system to explore the roles of gradients of extrinsic molecules in guiding neuronal axon growth.

The proposed research will provide an excellent opportunity for participating students to work at
one of the forefronts of nanoscience/technology, i.e., molecular nano-biology and nanobiotechnology. In addition to graduate training, the PIs plan to develop and co-teach a freshman seminar course annually on, "Nanobiology: science and engineering of neuron growth," to stimulate undergraduates' interest and participation. It is anticipated that their proposed activities will contribute to the preparation of future innovators and leaders in nanoscience in neurobiology and its practical application.