Ju-Ahng Lee, Ph.D. - US grants

DESCRIPTION (provided by applicant): Semaphorin pathways have been well characterized for their roles in axon guidance. Moreover, recent findings highlight significant roles of this pathway in other critical areas such as vasculogenesis, angiogenesis, cell migration/differentiation, immune regulation, and cancer pathology. The Pi's laboratory has recently identified zebra fish olfactomedin 2 (OM2), a secreted glycoprotein with a highly conserved olfactomedin domain. Initial expression and functional characterization studies revealed OM2's roles in three important developmental processes: axon guidance, neural crest migration/differentiation, and angiogenesis. Analyses on phenotypes of OM2 morphants in comparison with published phenotypes from disrupted semaphorin pathways, consistently led the PI to formulate the following overall hypothesis: OM2 regulates axon guidance, neural crest cell migration/differentiation, and angiogenesis via its interaction with the semaphorin pathway. In order to test the OM2-semaphorin pathway link in these processes, three Specific Aims are proposed. Specific Aim 1: To test the hypothesis that highly specific cranial axon guidance defects in OM2 morphants are due to the perturbation of direct interaction between OM2 and semaphorin receptor complexes. Specific Aim 2: To test the hypothesis that the absence of pharyngeal cartilages in OM2 morphants is due to the perturbation of cranial neural crest cell (cNCC) migration and/or differentiation, which is critically dependent upon semaphorin pathways. Specific Aim 3: To test the hypothesis that highly specific defects found only in late-onset cranial vasculature in OM2 morphants are due to perturbation in semaphorin-neuropilin and/or VEGF-neuropilin pathways. These hypotheses will be tested by (1) high resolution expression analysis of OM2 and semaphorin signaling molecules, deficiency of which results in highly similar phenotypes as OM2-deficiency phenotypes;(2) concomitant inhibition of semaphorin components and OM2 to verify functional convergence of OM2 in the semaphorin signaling pathway;(3) testing direct molecular interactions between OM2 and components of the semaphorin receptor complex. Given that the semaphorin pathway is known to be critically involved in adult nerve regeneration, craniofacial malformation (most frequent human birth defects), and tumor angiogenesis, novel insights into the regulation of this signaling pathway will be of paramount health benefit.

An award is made to North Carolina Central University (NCCU) to support the acquisition of a confocal microscope for research and training activities. The confocal microscope will be used extensively to support the fundamental research activities of twenty-eight tenure-track, independently funded faculty, each directing research programs that will significantly advance knowledge in their chosen fields and mentoring undergraduate and graduate students from predominantly minority communities. The new microscope will enable the training of the next generation of scientists in modern microscopy methods for the analysis of diverse biological samples. This system will be used to train students in state-of-the-art imaging acquisition applications via existing courses and during direct undergraduate and graduate research experiences. The microscope will also be incorporated into outreach activities for middle and high school students.

The new microscope will critically enhance the scientific findings of many NCCU faculty who study a diverse range of topics using zebrafish, mammalian cell culture, Drosophila, mice, and transgenic plants. Projects that will use the range of capabilities of the confocal microscope include: neuronal developing and patterning, recombinant protein expression in transgenic plants, analyses with multiple ligand/receptor targets, brain development and mitochondrial biogenesis, and regulation of cell growth. Together with 3D printing technologies, this state-of-the-art confocal microscope will serve as a key means to develop interdisciplinary collaborations between individuals in the biomedical sciences and engineering. Data and analyses from these studies will be published in peer-reviewed scientific journals, presented at scientific meetings, and used in both educational and public outreach activities.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

PROJECT SUMMARY/ABSTRACT Stroke is one of the leading causes of death in the U.S. and also a leading cause of adult disability with severe societal burdens. Ischemic strokes, caused by the thromboembolic occlusion of cerebral arteries, constitute the majority of stroke incidences and the rest are hemorrhagic strokes with ruptured blood vessels. In the U.S., stroke incidence shows clear disparity between African Americans and Caucasians with much higher frequency (~240%) in blacks. Despite intensive search for treatment, recombinant tissue plasminogen activator (rtPA) remains the only FDA-approved post-stroke medicine with limited effectiveness. Behind this woefully inadequate dearth of stroke therapeutics lies the difficulties in generating a sufficiently large number of stroke-induced animals for effective drug screening, as a highly labor-intensive surgical procedure (middle cerebral artery occlusion) is still the method of choice to induce ischemic strokes in model animals. Photothrombosis is one of rapidly adopted new methods to induce ischemic strokes in rodents, with a number of advantages such as highly reproducible infarct size and location with minimal mortality. In this procedure, focal illumination of defined wavelength light on the exposed skull activates an IV-injected photosensitive chemical (eg. Rose-Bengal) in the bloodstream, causing injuries in endothelial cells and local platelet aggregation, leading to the clogging of the affected blood vessel. However, even in this case labor-intensive procedures that cannot not easily be scaled up must be utilized to create the stroke model. Recently, photothrombic ischemic stroke has also been successfully induced in the adult zebrafish brain by focal illumination following a manual injection of Rose-Bengal, thus providing an additional vertebrate animal model system for stroke research. In this regard, an exciting novel genetic approach, which eliminates the manual injection of photosensitive chemicals, was recently created and tested successfully to selectively induce apoptotic cell death by light illumination in the adult zebrafish heart. Here we propose a creation of a readily scalable, optogenetically induced stroke system using transgenic zebrafish, development of a behavioral test system to identify novel therapeutic chemicals, and to test GWAS (genome wide association study)-identified stroke risk variants of the human APOL1 gene, also known as prominent kidney disorder risk factors in people of African ancestry.