Patricia E. Phelps - US grants

9734550 PHELPS Dr. Phelps' Early Career award contains both educational and research components related to the field of developmental neurobiology. The educational aim is to build an internet-based multimedia learning system for a new undergraduate course on developmental neurobiology, in collaboration with Dr. Gambhir and colleagues at the Crump Institute for Biological Imaging at UCLA. During nervous system development, many complex tissue movements occur that are difficult for students to understand using the typical 2-dimensional drawings from textbooks, yet they come alive when presented as 3-dimensional animations. Designing colorful animations depicting these complicated tissue movements is an important goal of this project. An additional focus is to incorporate data from scientists working in the field of developmental neurobiology. For example, time-lapse video microscopy demonstrating observations of living neurons will be used in classroom presentations to introduce students to exciting new research techniques. Students are able to review class presentations over the web, and utilize interactive quizzes as study aids. Such evaluation tools will help the students, but also will provide the teachers with information about the effectiveness of this new internet-based learning system. The aim of the research component is to determine what structures and/or molecules guide the unique migration of a group of chemically defined neurons during early spinal cord development. The neurons under study, called the "U-derived" cells, use acetylcholine as their neurotransmitter and can be identified by both immunological and histochemical methods. Between embryonic days 15 and 16, U-derived cells migrate from their initial ventral locations to their final destinations within the dorsal and central parts of the spinal cord. These cells migrate in a direction perpendicular to the radial pathways utilized by most other neurons. To determine the structural and/or molecular cues that guide this unique migration, Dr. Phelps will attempt to block the migration experimentally. Dr. Phelps uses an organotypic slice culture system that has been shown to support normal migration of U-derived cells and maintains relatively normal intercellular relationships between neurons and their environment, yet is accessible to both chemical and surgical manipulation, which would not be possible the whole embryo. A variety of different experiments are designed to disrupt this cell migration in culture. Results from these experiments will determine if the dorsal migration of the U-derived cells is guided by structural cues, such as early forming axons, and/or by molecular cues such as different types of adhesion molecules.

DESCRIPTION (provided by applicant): L1 is an axonal cell adhesion molecule (CAM) that is highly expressed on growing axons during development but decreases to low levels by adulthood. Ll CAM is highly conserved in mammals, and related molecules are found in diverse species, suggesting a conservation of its function in axonal pathfinding and fasciculation. L1 is expressed by olfactory ensheathing glia (OEG) and Schwann cells, both of which facilitate axon regeneration. In addition, Ll is re-expressed on regenerating axons in the hippocampal formation. Thus, Ll is an excellent candidate for an adhesion type molecule of critical importance to regenerating spinal cord axons. Specific Aim 1 is to determine the supraspinal target of a population of GABAergic commissural neurons that express Ll on the surface of their axons. Subsequent experiments will examine the axonal pathfinding of these commissural neurons in Ll knockout mice. Specific Aim 2 seeks to determine if Ll is re-expressed on adult axons as they regenerate new processes after spinal cord injury (SCI). Following a complete midthoracic spinal cord transection at postnatal day 5, we have preliminary data suggesting that Ll is re-expressed in axons both rostral and caudal to the lesion three months post injury. We will characterize the temporal expression of Ll on axons relative to the time post injury to provide information regarding potential axon sprouting after SCI. Furthermore, we will test if training spinal transected animals in spinal stepping patterns will change the level of Ll expression on lesioned axons. In Specific Aim 3, an OEG transplantation model will be used to determine if ascending sensory axons are able to project to their targets and if ascending and descending regenerating axons will re-express Ll CAM as they cross the transection site. Spinal transected animals will be transplanted with Ll-expressing OEG cells, whereas the controls will be injected with media. This OEG transplantation model has been shown by others to dramatically improve voluntary motor function in adult animals with SCI. Our long term goal is to develop a regeneration model for SCI that restores the ascending projections from commissural neurons.

Principal Investigator: Dr. Patricia E. Phelps
Title: Altered nociception and neuronal migration in the dorsal horn of reeler mice
Grant Number: IOB-0518714

The reeler mouse (a mouse whose reeler gene is defective) is characterized by motor coordination defects and tremors that result from errors in cell migration during development of the cerebral cortex and cerebellar cortex, both in the brain. Reelin, the protein missing in reeler mice, binds to lipoprotein receptors (VLDL/ApoE2), leading to phosphorylation of the intracellular protein Disabled-1 (Dab1). Mice missing either Dab1 or VLDL/ApoE2 receptors have anatomical and behavioral defects virtually identical to those of reeler mice, as these three molecules function in a common signaling pathway.

Dr. Phelps's lab has provided the first evidence that reeler mutants also have profound sensory defects, including mechanical insensitivity and increased thermal sensitivity. The first aim of this proposal is to determine if mice lacking Dab1 or VLDL/ApoE2 receptor also have defects in pain sensation. The second aim is to study the migratory errors that cause the abnormal sensory processing. Preliminary results suggest that part of the spinal cord (the superficial dorsal horn) in reeler mice contains mispositioned neurons. To prove that these migratory errors are caused by Reelin deficiency, the Reelin signaling pathway will be blocked with the goal of recapitulating the migratory errors in a culture dish. To further link the defects in pain processing with anatomical rearrangements, experiments will be conducted to determine which neurons along the pain processing pathway are responsible for the sensory defects. These studies will focus on the primary sensory neurons that detect pain and their targets in the dorsal horn of the spinal cord. Finally, the basic migratory patterns that establish dorsal horn lamination will be examined.

This proposal continues activities initially funded by an NSF Early Career Award. Integration of research and teaching activities includes teaching an undergraduate class on the principles of nervous system development and disseminating multimedia material developed for this class in the California Digital Library. Additionally, minority students have and will continue to conduct research in the Phelps' laboratory.

"This award is funded under the American Recovery and Reinvestment Act of 2009
(Public Law 111-5)."

Reelin, the protein missing in reeler mice, binds to lipoprotein receptors (VLDL/ApoE2) and causes phosphorylation of the intracellular protein Disabled-1 (Dab1). Mice with mutations in Reelin, VLDLR and ApoER2, or Dab1 have similar migratory errors in the superficial dorsal horn of the spinal cord, the termination site for primary sensory neurons that convey pain information into the central nervous system. Previous studies by the principal investigator's laboratory found a functional correlate of these developmental defects: an increased sensitivity to heat and a decreased sensitivity to mechanical pain. The differential effects observed on these pain modalities suggested that Reelin-signaling pathway mutants naturally segregate the biological basis of thermal and mechanical pain transmission.

The first aim of this project is to identify the mis-positioned neurons in the dorsal horn of Reelin-signaling pathway mutants using a "knock-in" mouse line with a blue reporter gene (beta-galactosidase) inserted in the Dab1 locus. Once the positioning errors in reeler and dab1 mutant dorsal horns are identified, they will use embryonic slice cultures to study the migratory defects in the mutant dorsal horn. The second aim will link the migratory errors to thermal nociceptive processing. Anatomical, behavioral, and perturbation experiments will determine if the heat hypersensitivity found in mutants is caused by mis-positioned neurons in the superficial dorsal horn that bear Neurokinin1 (NK1) receptors. Because the NK1 receptor is the target of the substance P-releasing nociceptors, it is expected that the ectopic NK1-expressing cells within the superficial dorsal horn are likely responsible for the heat hypersensitivity in Reelin-signaling pathway mutants.

Two undergraduate student researchers and a post-doctoral fellow will receive mentoring and research training as they conduct experiments related to this project. The broader impact will be to facilitate integration of research and teaching activities in developmental neurobiology and to support undergraduate and minority education and training in research careers.

DESCRIPTION (provided by applicant): Our two recent studies on the potential of olfactory ensheathing glial cell (OEC) transplantation provide conclusive evidence of functional re-connectivity and sensorimotor recovery in adult rats after a complete spinal cord transection. Based on these findings, the axon regeneration induced by OEC treatment facilitated some desired sensorimotor functions, but suppressed others. This proposal asks if the OEC effect on hindlimb motor function can be enhanced in both magnitude and specificity with different activity- based interventions. The central hypothesis is that the regenerative effects of OEC can be enhanced by activity-dependent mechanisms, such as epidural spinal cord stimulation (ES) combined with a serotonergic agonist, and training for a motor task (climbing or step training). Much work from our laboratory has focused on the effects of chronic, low intensity ES in completely paralyzed mammals, and although the mechanism is still unclear, ES plus the serotonergic agonist quipazine activates the lumbosacral neural circuitry and greatly enhances locomotion in spinal rats. Our recent work in a human model shows that ES, when combined with motor training, can trigger functional regenerative events and recovery of independent standing and volitional control of lower limb movements. To further develop strategies to amplify the magnitude of the OEC-mediated effects observed previously, two Specific Aims are proposed using fibroblast- and OEC-treated complete spinal cord transected rats and extensive electrophysiological, anatomical, and functional assessments. Specific Aim 1 will determine whether the regenerative effects of OEC transplantation are greater than those of the fibroblast controls, and if ES and quipazine (to modulate spinal excitability) or a voluntarily initiated training of a climbing task (to engage supraspinal pathways) will promote OEC-facilitated axonal regeneration and sensorimotor recovery. Specific Aim 2 asks if the regenerative effects of OEC transplantation are more robust when enhanced by the combined treatments of ES and voluntary climb training or the treatments of ES and treadmill step training. We anticipate that both the magnitude and specificity of the regeneration initiated by OEC transplantation will be most enhanced by ES and climb training, and that these interventions will stimulate the supraspinal and propriospinal networks to improve performance of selected sensorimotor tasks. Innovative features of these studies include the sophisticated measurements of evoked potentials in awake behaving spinal rats, a comprehensive battery of functional evaluation tools, and tracing experiments to detect regeneration of supraspinal and propriospinal neurons. The significance of these studies is to determine the extent to which both the amount of axon regeneration across the transection site and the specificity of the established re-connections can be enhanced by activity-dependent mechanisms. Ultimately, such mechanisms may be among the best candidates to enhance the functional benefits derived from OEC transplantation in completely paralyzed SCI patients. PUBLIC HEALTH RELEVANCE: Paralysis after a severe spinal cord injury (SCI) remains a major public health challenge due to the limited treatment options. Olfactory ensheathing cell (OEC) transplantation is a promising repair strategy that facilitates functional re-connectivity an improved sensorimotor function, but the magnitude and specificity of the effects must be enhanced. If one or more of the complementary activity-dependent treatments we propose to study, i.e., epidural spinal cord stimulation combined with a pharmacological stimulant, volitional motor training (climbing for a conditioned reward), and treadmill step training, significantly enhance the OEC effect, then these interventions could become viable, effective treatments for humans with severe SCI.