Gregory J. Cole - US grants

The neural cell adhesion molecule NCAM plays a critical role in mediating cell-cell interactions in the developing nervous system. Our laboratory has used monoclonal antibodies to characterize the structure-function relationship of NCAM, and to demonstrate for the first time that NCAM binds to heparan sulfate proteoglycan (HSPG). Monoclonal antibodies which recognize the heparan sulfate-binding domain of NCAM inhibit NCAM function, which provides strong evidence that HSPG participates in NCAM-mediated cell interactions during neural development. In the proposed study the neuronal HSPG(s) which interacts with NCAM will be identified and characterized. Conventional biochemical analysis of the HSPG will provide insight into the type of HSPG involved in cell-cell interactions in the developing nervous system. Neuronal HSPGs will be fractionated by virtue of the ability to co-purify with NCAM. Monoclonal and polyclonal antibodies will be produced which recognize the HSPG that binds to NCAM. This will permit the investigation of the role of HSPG in NCAM function using in vitro and in vivo functional assays. The antibodies will also be employed for he immunocytochemical analysis of the expression of the HSPG during neural development. The latter studies will provide information about the cellular distribution of a specific class of HSPG that is involved in NCAM-mediated cell adhesion. A second aim of the proposal is to examine the mechanism by which HSPG participates in NCAM function. Previous studies have employed lipid vesicles containing purified NCAM, in liposome aggregation assays, to demonstrate that NCAM mediates cell adhesion by a homophilic binding mechanism. In the proposed study we will examine whether HSPG co-purifies with NCAM because of its ability to bind specifically to NCAM. Liposome aggregation assays will then be conducted using purified NCAM (treated to remove HSPG) and HSPG to determine whether HSPG introduction into NCAM-containing liposomes alters NCAM binding affinity, and hence the rate of cell-cell adhesion.

Critical to the development of the nervous system is a series of events that depend upon cell recognition processes. These include the regulation of cell proliferation and differentiation, neuron migration, axonal outgrowth, and ultimately target cell recognition. The long-term goal of my laboratory has been to identify novel neural proteins that are expressed at these critical stages of neural development, and thus are likely to play an integral role in these cell recognition processes. Using monoclonal antibodies (MAbs) my laboratory has identified two novel neural proteins in the embryonic chick nervous system, which are associated with putative barrier structures in the CNS. Barrier structures have recently received increasing attention, since they may regulate axonal growth by inhibitory mechanisms. The two proteins identified in my laboratory are developmentally down-regulated during CNS development, and are not expressed in adult brain. The first protein, named Embryonal Avian Polypeptide of 300 kDa (EAP-300), has been shown to be restricted to dorsal midline structures in embryonic chick CNS, the glial knot barrier of the diencephalon, and is also expressed by radial glia in the developing cerebellum. The second molecule, a keratan sulfate proteoglycan (KSPG), was initially identified by its co-purification with EAP-300 under non-dissociating conditions. Antibodies to this KSPG stain the dorsal midline region of the chick CNS, suggesting that the association between these two molecules may have functional implications. It has been shown that the purified KSPG, named claustrin (from the Latin,claustra= barrier), inhibits cell adhesion on laminin or NCAM substrata. The studies proposed here will focus on a characterization of these two novel proteins, with an emphasis on their molecular and functional properties. The specific aims of this proposal are:1) to characterize these barrier-associated molecules, with an emphasis on their immunohistochemical distribution during development, an analysis of the possible functional interaction between claustrin and EAP-300, and the biochemical properties of the molecules; 2) to examine the function of EAP-300 and claustrin, and determine their role in the function of CNS barriers in situ; and 3) to isolate cDNAs coding for EAP-300 and claustrin, with an emphasis on the characterization of the developmental expression of EAP-300 and claustrin mRNA.

The long-term goal of this project is to obtain an understanding of the role of heparan sulfate proteoglycans (HSPGs) in the regulation of cell interactions in the developing nervous system. Previous studies have documented a critical role for HSPG in the function of the neural cell adhesion molecule (NCAM). In view of this importance we have recently begun to characterize the HSPG of developing chick brain that interacts with NCAM. Recent studies have demonstrated that an abundant developmentally-regulated HSPG in chick brain, which contains a protein core of 250 kDa, is capable of binding to the heparin-binding domain (HBD) of NCAM. The HSPG has been purified by monoclonal antibody affinity chromatography and shown to promote cell adhesion when used as a substratum. Its role in cell adhesion requires heparan sulfate chains on the HSPG, since elimination of heparan sulfate from the molecule abolishes its ability to participate in cell adhesion. In order to begin to understand the molecular mechanisms by which this HSPG regulates NCAM function and cell adhesion, we have initiated molecular cloning of the core protein of the HSPG. These studies indicate that the core protein is identical to agrin, an extracellular matrix protein suggested to play a critical role in neuromuscular synaptogenesis. These findings are of interest since the function of agrin in brain is unclear, and our data suggest that brain agrin, as a HSPG, may regulate cell adhesion processes. To continue our characterization of this HSPG, now named PG-agrin (proteoglycan-agrin), we will address the following specific aims: l) Conduct a molecular and biochemical characterization of PG-agrin, with an emphasis on determining if alternatively spliced variants of agrin encode PG-agrin. These experiments will also examine the relationship of agrin to PG-agrin, and determine if other brain HSPGs are capable of interacting with NCAM. 2) Determine the distribution of PG-agrin protein and mRNA in developing chick nervous tissue. 3) Examine the molecular mechanisms by which PG-agrin modulates NCAM function, using non-neural and neural cell lines transfected with NCAM and/or PG-agrin cDNAs. 4) Characterize the role of PG-agrin in chick brain development in vivo. PG-agrin sense and antisense plasmid cDNA will be introduced into developing chick embryos using lipospermine, and the effect of these plasmids on neural development will be examined. The proposed studies are important since they will ultimately provide insight into the function of HSPGs in neural development, and will also provide crucial information about possible functions of agrin during CNS development.

DESCRIPTION: (Applicant's Abstract) North Carolina Central University is responding to the NIH-National Institute on Drug Abuse and Office of Research on Minority Health initiative entitled HBCU Research Scientist Award. The proposed NCCU drug abuse research program in collaboration with Wake Forest University School of Medicine, will focus on molecular mechanisms of drug-cell interaction, seeking to understand the molecular correlates of addictive behavior. The program will be housed in the new Biomedical/Biotechnology Research Institute. Working with NIDA and ORMH, NCCU will address the program goals and support the growth and development of drug abuse research by: assembling a panel of scientists external to NCCU that could serve as a drug abuse research advisory committee: identifying and recruiting an experienced individual generally recognized as an accomplished drug abuse research scientist: establishing formal linkages with scientists at the drug abuse research center located at Wake Forest University School Medicine, located approximately 110 miles from NCCU: cost effective assembly and training of a high quality team of scientists to conduct drug abuse research at NCCU: and serving as a catalyst to enhance public awareness, particularly among the minority community, of the behavioral and public health consequences of drug addiction.

[unreadable] DESCRIPTION (provided by applicant): Recent studies have documented important roles for heparan sulfate proteoglycans (HSPGs) in aging and development of the nervous system. HSPGs are proposed to regulate processes as diverse as neural cell differentiation, cell adhesion, and the pathogenesis of human diseases such as Alzheimer's disease (AD). Recent studies from our laboratories have shown that agrin is a major extracellular matrix (ECM) and transmembrane HSPG in nervous tissue. Agrin is an extracellular matrix protein identified and named based on its involvement in the aggregation of acetylcholine receptors (AChRs) during synaptogenesis at the neuromuscular junction (NMJ). Emerging evidence indicates that agrin's function is not limited to its role in synaptogenesis, as agrin modulates axon extension, is expressed in adult brain, and may contribute to the etiology of some neurodegenerative diseases. The studies outlined in this proposal are aimed at understanding mechanisms by which agrin functions in brain development and aging. The specific goals of this proposal are: 1) To analyze the role of agrin in neurogenesis, neural patterning and axonal growth in the developing CMS, using in vivo approaches in chicken and zebrafish embryos. These studies will focus on analyzing the role agrin plays in modulating the function of heparin-binding signaling molecules such as the fibroblast growth factors (FGFs). Agrin morpholino oligonucleotides will be employed to knock-down agrin expression during chick or zebrafish development, and will be combined with inhibitors of FGF receptor function or FGF overexpression to explore agrin's role in the modulation of FGF signaling during development. 2) To elucidate the molecular mechanisms by which agrin regulates posterior development in zebrafish. These studies will test the hypothesis that agrin, via regulation of FGF signaling pathways, is necessary for posterior development in zebrafish. 3) To examine the role of agrin in the regulation of beta-amyloid aggregation, clearance and cytotoxicity in AD brain. These studies represent a continuation of our studies that suggest a crucial role for agrin in modulating beta-amyloid aggregation. These studies will include the use of AD mouse models to investigate further the role of agrin in AD. We predict that these proposed studies will provide important new insight into the function of agrin in the developing and aging nervous system. [unreadable] [unreadable]

Alcohol (ethanol) is a teratogen known to have diverse effects on brain and craniofacial development. Recent studies provide strong support for interference with Sonic hedgehog (Shh) signaling as a critical molecular event in Fetal Alcohol Spectrum Disorder (FASD). However, the mechanism for Shh interference is unknown and resulting abnormal phenotypes remain incompletely defined. For the current investigation, our overall goal is to determine whether ethanol-mediated interference with the function of key morphogenic proteins (especially Shh) in the extracellular matrix (ECM) underlies ethanol's teratogenicity, as well as documenting the dysmorphology that results from this interference. This innovative and novel work will focus on agrin, a major basement membrane and transmembrane HSPG for which our laboratory has extensive research experience. It will employ a model system that holds particular promise for identification of ethanol's teratogenic mechanisms, the developing zebrafish. Previous studies have shown that exposure of zebrafish embryos to ethanol results in brain and ocular dysmorphology, as well as behavioral defects. In previous studies, our laboratory has shown that loss of agrin function in zebrafish leads to microphthalmia. This appears to be mediated by disrupted Fgf and Shh signaling. Our preliminary studies also demonstrate that agrin gene expression in zebrafish eyes is diminished in response to ethanol exposure, and suggest that agrin knockdown and ethanol exposure may share a common pathway to produce ethanol-mediated ocular defects. Thus, the underlying hypothesis of this proposal is that agrin is a CNS target of embryonic ethanol exposure, with perturbed agrin function resulting in interference with normal Shh and/or Fgf signaling following ethanol exposure. This hypothesis will be tested in the following specific aims: 1) To test the hypothesis that CNS phenotypes induced in the zebrafish by embryonic ethanol exposure and agrin loss-of function are comparable; 2) To test the hypothesis that ethanol-induced diminution in agrin gene expression disrupts Shh and/or Fgf signaling, ultimately contributing to developmental abnormalities associated with FASD; and 3) To test the hypothesis that embryonic ethanol exposure in zebrafish disrupts GABAergic and dopaminergic neuron differentiation as a consequence of perturbed Shh, Fgf, and/or agrin function. Ultimately, these studies will begin to provide insight into the molecular basis of FASD, using zebrafish as a new model for the study of FASD.

DESCRIPTION (provided by applicant): This is an application for a cooperative agreement (U54), "Mechanisms of Alcoholic Pathology" (MAP), between faculties of the Biomedical/Biotechnology Research Institute (BBRI) at North Carolina Central University (NCCU), a Minority-Serving Institution, and the Bowles Center for Alcohol Studies at The University of North Carolina (UNC) School of Medicine. This proposal represents a true collaborative effort between the NCCU and UNC faculty with both groups contributing significant effort and being essential to the success of this CMARCD Program. Within this proposal, an NCCU Administrative component and three Research Components will integrate with the UNC-ARC Administrative, Education, 5 Research Components, and Cores, as well as other activities at the Bowles Center for Alcohol Studies. The UNC-ARC investigates mechanisms of alcohol pathology across the spectrum of behavioral, tissue, and cellular pathologies that occur with alcohol exposure. This proposal will focus on cellular pathologies that easily integrate into the ongoing overall theme of the UNC-ARC. Thus, both the UNC-ARC research components and this U54 proposal are integrated around the central theme that alcohol-induced pathology involves molecular and cellular changes that occur with alcohol abuse and alcoholism. The objectives of this U54 partnership are: 1) To investigate molecular mechanisms of alcohol-induced cellular pathology. By conducting an integrated and focused investigation into the molecular mechanisms of alcohol pathology this proposal will make important contributions to understanding alcohol morbidity and will create an active and successful research program on alcohol pathology at NCCU;2) To provide scholarly education on Alcohol Pathology. The educational efforts in this proposal will educate NCCU students on alcohol pharmacology and alcohol related pathologies and health disparities through a combined Annual Alcohol Research Day, new alcohol course curricula developed by NCCU faculty, an alcohol seminar series hosted by the BBRI, the UNC Bowles Center for Alcohol Studies seminar series, and NCCU fellowships to students on alcohol pathology and minorities. Ultimately, this proposal will conduct, promote, support, and mentor research into mechanisms of alcohol pathology, creating an acfive and successful alcohol research program within the NCCU-BBRI that synergizes with the UNC-ARC to advance education and discoveries.

Alcohol (ethanol) is a teratogen known to have diverse effects on brain and craniofacial development. Recent studies provide strong support for interference with Sonic hedgehog (Shh) signaling as a critical molecular event in Fetal Alcohol Spectrum Disorder (FASD). However, the mechanism for Shh interference is unknown and resulting abnormal phenotypes remain incompletely defined. For the current investigation, our overall goal is to determine whether ethanol-mediated interference with the function of key morphogenic proteins (especially Shh) in the extracellular matrix (ECM) underlies ethanol's teratogenicity, as well as documenting the dysmorphology that results from this interference. This innovative and novel work will focus on agrin, a major basement membrane and transmembrane HSPG for which our laboratory has extensive research experience. It will employ a model system that holds particular promise for identification of ethanol's teratogenic mechanisms, the developing zebrafish. Previous studies have shown that exposure of zebrafish embryos to ethanol results in brain and ocular dysmorphology, as well as behavioral defects. In previous studies, our laboratory has shown that loss of agrin function in zebrafish leads to microphthalmia. This appears to be mediated by disrupted Fgf and Shh signaling. Our preliminary studies also demonstrate that agrin gene expression in zebrafish eyes is diminished in response to ethanol exposure, and suggest that agrin knockdown and ethanol exposure may share a common pathway to produce ethanol-mediated ocular defects. Thus, the underlying hypothesis of this proposal is that agrin is a CNS target of embryonic ethanol exposure, with perturbed agrin function resulting in interference with normal Shh and/or Fgf signaling following ethanol exposure. This hypothesis will be tested in the following specific aims: 1) To test the hypothesis that CNS phenotypes induced in the zebrafish by embryonic ethanol exposure and agrin loss-of-function are comparable; 2) To test the hypothesis that ethanol-induced diminution in agrin gene expression disrupts Shh and/or Fgf signaling, ultimately contributing to developmental abnormalities associated with FASD; and 3) To test the hypothesis that embryonic ethanol exposure in zebrafish disrupts GABAergic and dopaminergic neuron differentiation as a consequence of perturbed Shh, Fgf, and/or agrin function. Ultimately, these studies will begin to provide insight into the molecular basis of FASD, using zebrafish as a new model for the study of FASD.

This proposal for a cooperative agreement will establish collaborative partnerships between the UNC-Bowles Center for Alcohol Studies (UNC-ARC) and NCCU-BBRI faculty. The program administrators of the National Institute on Alcohol Abuse and Alcoholism will be involved in steering the program. These partnerships were developed to perfectly fit the long-range goal of the Collaborative Minority Alcohol Research Center Development (CMARCD) Program. That is to strengthen the alcohol research capacity of minority serving institutions. These collaborations are structured to integrate research between laboratories leading to common collaborative discoveries, with the overall objective to strengthen alcohol research programs at both institutions. This proposal represents a true collaborative effort between NCCU and UNC faculty with both groups contributing significant effort and being essential to the success of this CMARCD Program to conduct research on the Mechanisms of Alcohol Pathology (MAP). The MAP-Administrative Core (MAP-AC) is responsible for the overall implementation, management, coordination and support of the jointly developed research components (Figure 1), as well as coordination with the UNC-ARC's Administrative Core. The objective is to ensure a cohesive seamless administration of the overall partnership. This cohesiveness is essential to the fulfillment of the partnership's goals and objectives. The overall coordination, planning and evaluation ofthe partnership will be through the Steering Committee (SC). The SC will advise the MAP-AC on implementation, coordination and management ofthe cooperative agreement program. The administrative core of this cooperative agreement is organized to manage, direct and coordinate research, training and outreach-education activities. The Administrative core will provide leadership for the conduct of competitive alcohol research at NCCU in collaboration with UNC-ARC. The administrative core will support the Co-Directors in their goals to integrate research themes, promote collaborations, introduce new methodologies and help train and advise individuals on research techniques, career paths, productive experimental designs and aspects of publication and review. It will be a catalytic stimulus for enhancing and promoting alcohol research at NCCU in collaboration with the experienced UNC-ARC faculty. The full potential of this mutually beneficial CMARCD partnership between NCCU and UNC will be realized through two Specific Aims: 1. To investigate molecular mechanisms of alcohol-induced cellular pathologv. This proposal will connect the UNC-ARC and NCCU faculty research components in collaborative partnerships focused on defining molecular mechanisms involved in alcohol pathology. By design, each research component will examine molecular processes leading to cellular pathologies, specifically fetal neurotoxicity, adult brain stem cells, and squamous cell carcinomas associated with alcohol abuse. By conducting an integrated and focused investigation into the molecular mechanisms of alcohol pathology this proposal will make important contributions to understanding alcohol morbidity and will create an active and successful research program on alcohol pathology at NCCU. 2. To provide scholarly education on Alcohol Pathology. The UNC-ARC education programs lead the state in alcohol CME for health professionals, as well as training medical and graduate students on alcohol related pathology. NCCU faculty and students will be exposed to these education programs, as well as a wide variety of alcohol research seminars, laboratory meetings, research methodologies, techniques, unique equipment and unique models of alcohol pathology at UNC. The alcohol educational efforts in this proposal will involve NCCU graduate students through a combined Alcohol Annual Research Day, new course curricula on alcohol pathology developed by NCCU faculty, an alcohol seminar series hosted by the BBRI, and the UNC-ARC biweekly alcohol seminar series, as well as specific undergraduate student NCCU Disparity fellowships to students to study and learn about alcohol pathology and health disparities. This proposal will develop alcohol research expertise and promote alcohol research infrastructure at NCCU, an MSI. Further it will educate, involve and develop researchers from underrepresented populations through recruitment of NCCU students. Undergraduate Disparity Fellowships and alcohol curriculum development will help identify and involve individuals in learning about and reducing alcohol-related health disparities. The Specific Aims ofthis CMARCD partnership will be achieved via six goals ofthe Administrative Core: 1. Provide scientific and administrative leadership for development and maintenance of the MAP-AC. 2. Maintain and enhance the partnership between the UNC Bowles Center and the NCCU/BBRI. 3. Recruit, select and mentor undergraduate and graduate students to understand the causes and consequences of alcohol use disorders and to participate in the conduct of alcohol research. 4. Recruit new or existing faculty at NCCU to expand their research interests to include alcohol research. 5. Organize an Annual Research Day of NCCU and UNC researchers that precedes the annual RSA meeting, providing a forum for NCCU faculty and students to present their alcohol research progress. 6. Evaluation of CMARCD progress with measurable objectives.

This proposal for a cooperative agreement will establish collaborative partnerships between the UNC-Bowles Center for Alcohol Studies (UNC-ARC) and NCCU-BBRI faculty. The program administrators of the National Institute on Alcohol Abuse and Alcoholism will be involved in steering the program. These partnerships were developed to perfectly fit the long-range goal of the Collaborative Minority Alcohol Research Center Development (CMARCD) Program. That is to strengthen the alcohol research capacity of minority serving institutions. These collaborations are structured to integrate research between laboratories leading to common collaborative discoveries, with the overall objective to strengthen alcohol research programs at both institutions. This proposal represents a true collaborative effort between NCCU and UNC faculty with both groups contributing significant effort and being essential to the success of this CMARCD Program to conduct research on the Mechanisms of Alcohol Pathology (MAP). The MAP-Administrative Core (MAP-AC) is responsible for the overall implementation, management, coordination and support of the jointly developed research components (Figure 1), as well as coordination with the UNC-ARC's Administrative Core. The objective is to ensure a cohesive seamless administration of the overall partnership. This cohesiveness is essential to the fulfillment of the partnership's goals and objectives. The overall coordination, planning and evaluation of the partnership will be through the Steering Committee (SC). The SC will advise the MAP-AC on implementation, coordination and management of the cooperative agreement program. The administrative core of this cooperative agreement is organized to manage, direct and coordinate research, training and outreach-education activities. The Administrative core will provide leadership for the conduct of competitive alcohol research at NCCU in collaboration with UNC-ARC. The administrative core will support the Co-Directors in their goals to integrate research themes, promote collaborations, introduce new methodologies and help train and advise individuals on research techniques, career paths, productive experimental designs and aspects of publication and review. It will be a catalytic stimulus for enhancing and promoting alcohol research at NCCU in collaboration with the experienced UNC-ARC faculty.

DESCRIPTION (provided by applicant): This is an application for a cooperative agreement (U54), Mechanisms of Alcoholic Pathology (MAP), between faculties of the Biomedical/Biotechnology Research Institute (BBRI) at North Carolina Central University (NCCU), a Minority-Serving Institution, and the Bowles Center for Alcohol Studies at The University of North Carolina (UNC) School of Medicine. This proposal represents a true collaborative effort between the NCCU and UNC faculty with both groups contributing significant effort and being essential to the success of this CMARCD Program. Within this proposal, an NCCU Administrative component and three Research Components will integrate with the UNC-ARC Administrative, Education, 5 Research Components, and Cores, as well as other activities at the Bowles Center for Alcohol Studies. The UNC-ARC investigates mechanisms of alcohol pathology across the spectrum of behavioral, tissue, and cellular pathologies that occur with alcohol exposure. This proposal will focus on cellular pathologies that easily integrate into the ongoing overall theme of the UNC-ARC. Thus, both the UNC-ARC research components and this U54 proposal are integrated around the central theme that alcohol-induced pathology involves molecular and cellular changes that occur with alcohol abuse and alcoholism. The objectives of this U54 partnership are: 1) To investigate molecular mechanisms of alcohol-induced cellular pathology. By conducting an integrated and focused investigation into the molecular mechanisms of alcohol pathology this proposal will make important contributions to understanding alcohol morbidity and will create an active and successful research program on alcohol pathology at NCCU; 2) To provide scholarly education on Alcohol Pathology. The educational efforts in this proposal will educate NCCU students on alcohol pharmacology and alcohol related pathologies and health disparities through a combined Annual Alcohol Research Day, new alcohol course curricula developed by NCCU faculty, an alcohol seminar series hosted by the BBRI, the UNC Bowles Center for Alcohol Studies seminar series, and NCCU fellowships to students on alcohol pathology and minorities. Ultimately, this proposal will conduct, promote, support, and mentor research into mechanisms of alcohol pathology, creating an acfive and successful alcohol research program within the NCCU-BBRI that synergizes with the UNC-ARC to advance education and discoveries.

? DESCRIPTION (provided by applicant): This is a renewal application for a cooperative agreement (U54), Mechanisms of Alcohol Pathology (MAP), between faculties of North Carolina Central University (NCCU), a Historically Black College and University, and the Bowles Center for Alcohol Studies (BCAS) at The University of North Carolina (UNC) School of Medicine. This proposal represents a true collaborative effort between the NCCU and UNC faculty with both groups contributing significant effort and being essential to the success of this Alcohol and Health Disparity Research Center (AHDRC) Program. Within this proposal, an NCCU Administrative Core, Mentoring and Development Core, and five Research Components will integrate with the UNC-BCAS Administrative, Mentoring and Development Cores and Research Components, as well as other activities at the Bowles Center for Alcohol Studies. The UNC NIAAA Alcohol Research Center (ARC) and BCAS have many faculty studying mechanisms of alcohol pathology across the spectrum of behavioral, tissue, and cellular pathologies that occur with alcohol exposure. This proposal will focus on cellular pathologies that allow collaborative research partnerships on common areas of interest between UNC and NCCU faculty. These partnerships open UNC research laboratories, core facilities and libraries to NCCU faculty improving opportunities for developing productive research laboratories at NCCU. In addition, large numbers of NCCU students will continue to become involved in research on mechanisms of alcohol pathologies providing education on African-American alcohol related health disparities which are due to increased morbidity to alcohol related pathologies and not increased alcohol use disorder. The objectives of this U54 partnership are to investigate molecular mechanisms of alcohol- induced cellular pathology. This proposal includes eight NCCU research projects that will investigate the molecular mechanisms of fetal alcohol pathology, alcohol induced liver disease, alcohol induced oral and breast cancers as well as alcohol-cannabinoid neurotoxicity. A second objective is to provide scholarly education on Alcohol Pathology for NCCU students. The educational efforts in this proposal will involve NCCU undergraduate and graduate students through the BCAS seminar series, through training in UNC laboratories and research development and mentor meetings, attendance and presentation of data at the annual RSA meeting, an alcohol seminar series hosted by NCCU, a new course curricula on alcohol pathology developed with a focus on health disparities, as well as specific undergraduate student summer internships for NCCU students to study and learn about alcohol pathology and health disparities within laboratories in both institutions. Ultimately this proposal will conduct, promote, support, and mentor research into mechanisms of alcohol pathology, creating an active and successful alcohol research program within NCCU that synergizes with UNC to advance education and discoveries.

This Administrative Core (U54), ?Mechanisms of Alcohol Pathology? (MAP), is a partnership between the Biomedical/Biotechnology Research Institute (BBRI) and Biomanufacturing Research Institute and Technology Enterprise (BRITE) institutes at North Carolina Central University (NCCU), a Historically Black College and University, and The University of North Carolina School of Medicine Bowles Center for Alcohol Studies (UNC-BCAS). Collaborative partnerships between these groups initated alcohol research at NCCU during the previous funded period with 3 active laboratories that have produced about a dozen publications, have involved 15-20 NCCU students per year in alcohol research with faculty submitting several NIH grants. This proposal continues to expand alcohol research to 8 active faculty alcohol research laboratories. This proposal includes an Administrative core, Mentoring and Development Core, and five Research Components, two full projects and 3 pilot projects, all with preliminary data and structured collaborative partnerships between NCCU and UNC-BCAS faculty. This U54 Mechanisms of Alcohol Pathology has research projects focused on fetal alcohol spectrum disorders, alcoholic liver disease, alcohol induced oral and breast cancers and alcohol-cannabinoid neurotoxicity. Health disparities in African-Americans is related to increased alcohol pathology, and not increased alcohol use disorder. These projects address alcohol health disparities by involving NCCU students in alcohol research that educates them about mechanisms of alcohol pathology associated with health disparities as well as building on the expertise of NCCU faculty. UNC collaborators chosen for appropriate expertise in the scientific area and include senior mentors who provide leadership, experimental design and scientific expertise as well as junior mentors who can help with hands on methods mentoring. Thus, UNC and NCCU this are integrated around the central theme of alcohol-induced pathology promoting molecular and cellular research involving NCCU faculty and students. The objectives of this U54 Administrative Core are: 1) Provide scientific and administrative leadership by advocating with leadership for staff support of research as well as supporting specific staff for alcohol research. By assuring UNC mentors collaborate and meet regularly with NCCU faculty and by organizing seminars, and annual steering committee meeting to review progress with NIAAA staff. NCCU faculty and students will do experiments in UNC laboratories, and gain access to UNC research cores and libraries. Research laboratory experience, access to full text journals and key research core facilities will improve It will also mentor NCCU undergraduate and graduate students training future alcohol researchers. Finally, the U54 Administrative Core will define expectations and metrics for assessment of progress. The MAP Administrative core will provide support that will allow NCCU faculty to develop independent self-supporting research programs at NCCU that will involve many students in AHDRC.

This U54 component, Research Project 3-NCCU-UNC Small Projects, will support the research development of three NCCU investigators new to alcohol research. These three small projects will receive funding to obtain pilot data that will allow the submission of grant applications to transition to independent funded alcohol research programs, or the transition to full research projects in later years of this U54 program. Through active mentoring received from the Mentoring and Development Core these NCCU alcohol researchers will further the understanding of the mechanisms of alcohol pathology as related to the role of alcohol exposure during development on microglia function, the role of alcohol on mammary tumor development, and the role of alcohol in pulmonary inflammation. It is expected that these pilot projects will introduce underrepresented students to alcohol research while enhancing the alcohol research expertise of the NCCU faculty. Ultimately these three pilot projects will continue the expansion of alcohol research at an IUCP, NCCU, and will advance our understanding of the contribution of alcohol abuse to cancer, lung inflammation, and the role of microglia in FASD.

This U54 Mentoring/Development Core: Mechanisms of alcohol pathology proposal will continue and expand mentoring and collaborative partnerships between the UNC-Bowles Center for Alcohol Studies (UNC-BCAS) and NCCU faculty and students that over the past 4 years has involved 8 NCCU Biomedical faculty, as many as 15 students each year, 12 manuscripts, and multiple alcohol NIH grant applications, none of which occurred prior to this partnership. This partnership has been extremely productive in initiating alcohol research at NCCU, a Historically Black College and University (HBCU) where 81% of students are underrepresented minorities (African-American or Hispanic). It continues to expose large numbers of underrepresented minority students to alcohol research to inform them regarding alcohol related pathology. The Mentoring and Research Development Core will support UNC faculty mentors providing research expertise to educate, involve and develop faculty and student researchers from NCCU through productive research collaboration and mentoring. The Mentoring/Development Core will provide funds for two years to maintain NCCU essential research staff in the 3 active laboratories with the goal of obtaining independent research support within this period. UNC mentors are supported for active NCCU programs as well as future new programs. By conducting an integrated and focused investigation into the genetic and molecular mechanisms of alcohol pathology this proposal will make important contributions to understanding alcohol morbidity and will create successful research programs exposing large numbers of NCCU students to alcohol research and alcohol pathology. Blacks are at high risk for fetal alcohol syndrome (FAS), alcoholic liver disease, and mortality from cancer of the oral cavity, pharynx, larynx, and esophagus. Undergraduate research internships and alcohol curriculum development will help identify and involve individuals in learning about and reducing alcohol-related health issues. Active NCCU alcohol research laboratories are training students from diverse backgrounds and are uniquely positioned to engage in research and in the translation of research advances into culturally appropriate education and health care.

It is increasingly clear that proteins of the extracellular matrix (ECM) are key regulators of cell patterning and morphogenesis in the developing nervous system. Growth factors and morphogens, such as fibroblast growth factors (Fgfs), sonic hedgehog (Shh) and retinoic acid (RA), are critical regulators of nervous system development. The present proposal continues our analysis of the effects of ethanol exposure during embryogenesis on ECM function, in particular agrin Shh, Fgfs and RA. Our overall goal is to understand how gene-ethanol interactions contribute to the hallmark features of fetal alcohol spectrum disorders (FASD). A focus of this proposal is to employ a new multiple binge-like alcohol exposure model during defined periods of zebrafish and mouse development to identify critical stages that are sensitive to ethanol exposure and that lead to the morphological and behavioral changes associated with FASD. The zebrafish model offers a unique advantage in that following ethanol removal the tissue levels of ethanol rapidly return to control levels, allowing defined exposures during embryogenesis. Thus, our underlying hypothesis is that gene-ethanol interactions involving agrin, Fgfs, Shh and RA during defined stages of embryogenesis are necessary to produce the hallmark morphological features of FASD, and the behavioral deficits that are characteristic of FASD. Our proposed studies take the innovative approach of using multiple subthreshold binge ethanol exposures during embryogenesis to reconcile the disparity between reported alcohol use during pregnancy and the incidence of FAS or FASD in the population. The specific aims of this proposal are: 1) To test the hypothesis that multiple binge-like alcohol exposures during defined stages of zebrafish or mouse embryogenesis produce characteristic morphological FASD phenotypes and associated gene expression alterations; 2) To test the hypothesis that multiple binge-like alcohol exposures during defined stages of zebrafish or mouse embryogenesis lead to behavioral changes associated with FASD in humans. These Specific Aims will further establish that pathological and behavioral phenotypes associated with FASD are regulated by the timing of ethanol exposure and the underlying gene-ethanol interaction, and will elucidate the role of ECM ligands such as agrin, Shh, Fgfs and RA in the ethanol-induced morphological and behavioral defects associated with these binge-like alcohol exposures.