Rosa E. Blanco - US grants

bioimaging /biomedical imaging; histochemistry /cytochemistry

cytoprotection; nerve injury; growth factor receptors; optic nerve; eye injury; cell growth regulation; retinal ganglion; neural plasticity; BCL2 gene /protein; protein kinase C; axon; biological signal transduction; eye regeneration; gene expression; fibroblast growth factor; mitogen activated protein kinase; active sites; gel electrophoresis; immunocytochemistry; western blottings; polymerase chain reaction; Anura; electron microscopy; in situ hybridization;

In most higher animals, injury to axons within the central nervous system (CNS) leads to the death of axotomized neurons, probably due to loss of target-derived growth factors. The long-term goal of this research is to understand the mechanisms by which growth factors promote the survival of axotomized ganglion cells in the retina. Due to its great accessibility, and ability to regenerate, the frog visual system is an excellent model with which to address this question. After lesioning the frog optic nerve, regeneration occurs and retinal ganglion cells (RGCs) reconnect to their targets. Nevertheless, approximately 50% of the retinal ganglion cells die. We have shown that application of fibroblast growth factor-2 (FGF-2) prevents this cell death in the retina, but only when it is applied to the optic nerve stump. There is accumulating evidence from in vitro studies that the signaling pathways activated by neurotrophins differ based on the location of stimulation, but little is known about how this site-specificity affects the responses to neurotrophins in vivo. In this proposal, we will determine the different intracellular signaling pathways that are activated by the neurotrophic factor FGF-2 when it is applied to the cut nerve, compared to intraocular application, that may explain its location-specific effects on neuronal survival. In the first aim we will test the hypothesis that FGF-2 applied to the cut axons, but not to the cell bodies, activates the transcription factor CREB via the MAPK pathway, in particular via Erk1/2. In the second aim we will determine the mechanisms by which FGF-2 upregulates the synthesis of brain-derived neurotrophic factor (BDNF), another neurotrophin essential for retinal ganglion cell survival, and of the BDNF receptor, TrkB. Finally, in the third aim we will investigate the location-specific effects and signaling pathways of BDNF itself, and determine how the site of application of the factor affects retinal ganglion cell survival. The proposed research will make a significant contribution towards understanding how neurotrophins modulate neuronal survival in a location-specific manner, in an in vivo adult system, which should lead to improved therapeutic methods to promote recovery after injury and disease.

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

Funds from the Major Research Instrumentation-Recovery and Reinvestment (MRI-R2) program were provided to support the acquisition of a new transmission electron microscope (TEM) to replace a 30 year old TEM at the University of Puerto Rico Medical Sciences Campus (UPR-MSC). The TEM will be installed at the Institute of Neurobiology. The Institute is an interdisciplinary, interdepartmental research facility devoted to the study of the structure and function of the nervous system at all levels of organization from whole animal behavior to molecular biology. Because it is managed as a shared resource, the new TEM significantly enhances the research efforts of investigators at the Institute, at other campuses of the UPR, and at other universities in Puerto Rico. The projects benefitting from the TEM address the broad objectives of understanding basic mechanisms of connectivity of the nervous system, focusing on changes during development, after injury, and in response to the environment. The acquisition of the TEM also positively impacts training of graduates and medical students, undergraduates and science teachers. Courses in basic and advanced electron microscopy enrich graduate programs and increase the number of qualified users on the island. Community outreach efforts, which include workshops in electron microscopy for high school teachers and visits to the new facility by groups of high school students, also broaden the impact of the instrumentation upon science education. Following in the tradition of the UPR as a major contributor to the education and training of scientists, the shared electron microscope is instrumental in the training of future academic leaders in the field of Neuroscience. Results from the studies enabled by the new TEM will be disseminated by student and faculty presentations at regional and national meetings, and through publication in peer-reviewed journals.

DESCRIPTION (provided by applicant): Abstract After lesioning the frog optic nerve, regeneration occurs and retinal ganglion cells (RGCs) reconnect to their targets. Even so, approximately 50% of the retinal ganglion cells die, compared to mammals where almost all do so. Application of neurotrophins such as brain-derived neurotrophic factor (BDNF) can largely prevent this cell death. However, although these factors are known to have potent effects on axonal growth, branching and synapse formation during development, very little is known about how they will affect regrowing axons during regeneration and reconnection to the target. Before neurotrophins can be used therapeutically to increase ganglion cell survival it is important that these potential effects on target reconnection are understood. The following aims should help in that understanding, and will contribute to our long-term goal of elucidating the molecular requirements for achieving complete and accurate regeneration of the optic pathway. In the first aim, we will test the hypothesis that axonally-applied BDNF increases the speed of axonal regeneration. This will be tested using assays of axonal growth with immunohistochemical markers, and by investigating whether blockers of the Erk and PI3K signaling pathways slow axonal regeneration. In the second aim, we will test the hypothesis that BDNF is synthesized in the tectum, and that increasing tectal BDNF levels promotes RGC branching and synapse formation and modulates the refinement of the retinotopic projection during regeneration. In the third aim, we will investigate how the levels and patterns of visual activity affect the synthesis of BDNF by regenerating frog RGCs, and whether these changes in BDNF synthesis in RGCs in turn affect the refinement of the regenerating retinal projection. This research will help our understanding of the molecular pathways involved in the regrowth and reconnection of regenerating nerve cells in the central nervous system. PUBLIC HEALTH RELEVANCE: Relevance This research will help our understanding of the molecular pathways involved in the regrowth and reconnection of regenerating nerve cells in the central nervous system. This basic knowledge will increase our understanding of the molecular mechanisms involved in the formation and modulation of synaptic connections during regeneration. It will also have the potential to contribute towards improved therapeutic methods to promote recovery after injury and disease.

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

Founded as an Institute within the University of Puerto Rico in 1967 by the renowned neuroscientist José del Castillo, the Institute of Neurobiology (INB) was established with the goal of using simple organisms to understand neural structure and function. UPR is a major minority-serving institution, and the INB graduate student population is predominantly Hispanic. The focus on Poikliothermic model systems holds new significance in furthering the understanding of the impact of climate change (seawater temperature, salinity, etc.) on ecosystems. The institution is focusing on simple organisms, and common interests such as neural plasticity, temperature adaptation, and signaling molecules. Funds are provided to correct significant deficiencies including 1) an antiquated air conditioning system and 2) an obsolete network cyberinfrastructure. The rejuvenation of the INB will significantly improve ongoing and planned research opportunities and create new opportunities for collaborative research. The renovations will have immediate broader impacts including: 1) significant benefit to the minority student body served by the University of Puerto Rico; 2) an innovative approach to "ecological neurobiology" that will provide the first wave of researchers equipped to deal with the emerging and important issues of global climate change; 3) the establishment of a Neurobiology Education Center that will serve to educate teachers, children and the public about the study of neuroscience, and the special relevance of these studies to Puerto Rico's tropical ecosystem.

? DESCRIPTION (provided by applicant): Effects of Retinoic Acid Signaling on Retinal Ganglion Cell Survival and Regeneration. Recovery of vision after optic nerve injury requires retinal ganglion cell (RGC) survival, axonal regrowth past the area of the lesion and reformation of appropriate synaptic targets. The adult mammalian visual system regenerates poorly, and past therapeutic efforts have enabled regrowth of only a small number of retinal axons. Frog RGCs, on the other hand, suffer an approximately 50% cell loss after injury, but regeneration and reconnection to target areas still occur because of the lack of inhibitory glial molecules and physical obstructions. This makes them a good model to explore which molecules influence RGC regeneration. Understanding what occurs after RGC injury is of great medical importance, because it results in blindness after optic nerve injury, and also in conditions such as glaucoma, diabetes and optic ischemia. The focus of the proposed work is to understand how the activity of retinoic acid (RA) signaling, known to exert effects on neurite outgrowth and homeostatic synaptic plasticity in the brain, affects the processes leading to recovery of vision after axotomy in the adult visual system. The aims of this proposal are to (1) determine the role of RA and the intracellular signaling pathways it activates in promoting RGC survival after injury, (2) determine the role of RA promoting regeneration of RGC axons, and (3) investigate the effects of RA on the reformation of synaptic connections in the retinorecipient layer of the optic tectum. The first aim will be accomplished by quantification of numbers of surviving RGCs, and Western blot analysis of regenerating retinal tissue. The second aim will be addressed by measuring the number of viable retinal axons crossing the lesion area, changes in the speed of regeneration, and changes in the abundance of GAP43 (regeneration marker) and NAV-2 (responsive gene in RA-mediated neurite outgrowth) in non-treated optic nerves and nerves from animals treated with RA signaling agonists and antagonists. The effect of RA signaling on the activation of relevant intracellular signaling pathways in regenerating RGCs will also be studied. The third aim will be carried out by quantifying anterogradely labeled retinal axons, the number of functional synaptic inputs, and the distribution of synaptic proteins in the retinorecipient layer f optic tectum after altering retinoic acid signaling and axotomy. Understanding the role of RA signaling in the response of retinal ganglion cells to nerve injury is important for the design of potential new therapeutic strategies.