Rachael L. French - US grants

[unreadable] DESCRIPTION (provided by applicant): Alcoholism is a common disease, affecting 4-5% of the population (reviewed by Zernig et al., 1997). In 1990, the cost of alcoholism and alcohol abuse to U.S. society was estimated at $136 billion (reviewed by Diamond, 1992). Despite these facts, our understanding of how alcohol affects behavior and brain function is incomplete. This proposal describes the identification of a mutation (EP1455) in a Drosophila MAPKKK gene, which causes an altered behavioral response to ethanol, cocaine, and nicotine. MAPK signaling is altered upon exposure to chronic ethanol in both rats and human hepatocyte cell culture (Chen et al., 1998; Kishore et al., 2002; Nelson et al., 2003), indicating that MAPK pathways are likely important targets for ethanol's effects on the brain. The goal of this project is to use EP1455 and the abundant genetic tools available in Drosophila to investigate the role of MAPK signaling in the response to ethanol. The specific aims of this proposal are: [unreadable] 1. Determine the spatial and temporal requirements for the MAPKKK encoded by CG14217. [unreadable] 2. Characterize the functions of the kinase and PERM domains in the function of CG14217. [unreadable] 3. Investigate the function of MAP kinase signaling in drug response. [unreadable] [unreadable]

Funds from this Major Research Instrumentation Program grant will be used for the purchase and support of a Zeiss LSM 700 Confocal Microscope at San José State University. Research groups from the Biology, Chemistry and Civil Engineering departments will be among the major users, as well as a biology professor from Santa Clara University. A common theme to the research that will be enabled and enhanced by this acquisition is to elucidate biomechanistic processes. Faculty members with expertise in the areas of genetics, development, environmental microbiology, cell biology, immunology, biochemistry, and civil engineering will comprise the major users of the confocal microscope. Specifically, the following research projects will utilize this microscope: apoptotic induction by r-disintegrins; molecular mechanisms that mediate neuronal circuit formation and maintenance; mechanisms by which ethanol exposure affects synaptic plasticity; characterization of novel uncultured bacterial groups for ecophysiological studies; immunomodulation of lymphocyte trafficking; cellular localization of inositol glycans; examining angiogenesis during ovarian development in mice, signal transduction resulting from binding of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] to the nuclear vitamin D receptor (VDR); using microbiological dating and confocal microscopy to date structural cracks; and developmental analysis of LIN-1 expression.

San José State University is a minority serving institution in Northern California with a large proportion of students with physical disabilities. Research groups actively incorporate undergraduate researchers and students at the Master?s level in their projects. Four main components of the training infrastructure at the university will be impacted by the purchase of this confocal microscope: 1) increased involvement of undergraduate students in research, 2) promoting active learning, 3) student training in specialized masters programs, and 4) collaborations with faculty and students at other institutions. Faculty that will utilize this confocal microscope are research mentors for several programs that aim to increase the number of minority students who pursue Ph.D. degrees in the sciences, including NIH-MARC, MBRS-RISE, HHMI-SCRIBE, and NSF-REU RUMBA. Learning to use this state of the art microscope in their research will help prepare students for careers in science. In addition, data collected from this microscope will be used in undergraduate courses to bring contemporary research into the classroom. Finally, smaller institutions in the surrounding community including Santa Clara University will have the opportunity to utilize this microscopy to increase their research potential.

DESCRIPTION (provided by applicant): Prenatal exposure to ethanol in mammals leads to a range of developmental problems, from growth deficiency to mental retardation and behavioral abnormalities. In humans, these symptoms are collectively described as fetal alcohol syndrome (FAS). Ethanol exposure is especially damaging to the developing nervous system and this has long-term consequences on adult behavior. The toxicity of developmental ethanol exposure has been attributed to numerous mechanisms, including ethanol metabolism and related oxidative stress, neuronal cell loss, and inhibition of growth factors and/or their signal transduction pathways. Finally, while human epidemiological data, twin studies, and animal models indicate that genetic factors confer risk for and protection from fetal alcohol injury, no genes altering susceptibility to FAS have been conclusively identified. The goal of our research is to identify and study the molecular targets of developmental ethanol using the genetically amenable model organism Drosophila melanogaster. Drosophila melanogaster, the common fruit fly, has been utilized extensively in biological research, particularly in genetics and development. Drosophila are particularly amenable to sophisticated genetic analyses, including genomic approaches, reverse and molecular genetics, and traditional forward genetic screens. Moreover, over a century of research has led to an extensive collection of genomic, molecular and genetic tools, making Drosophila tremendously powerful in the elucidation of gene function. We have developed a genetic model of FAS in flies. We have shown that developmental ethanol exposure causes reduced viability and growth delay. In addition, as in mammals, flies reared on ethanol have altered behavioral responses to ethanol intoxication as adults. Finally, we have found that the developmental and behavioral defects are due to ethanol's effects on insulin signaling, as well as effects on the epidermal growth factor receptor (EgfR) pathway. Our research will further elucidate the role of insulin signaling in the development of FAS, as well as identify additional genetic and cellular targets of developmental ethanol exposure, both downstream and independent of insulin signaling. Our specific aims are: 1) to determine the role of the EgfR pathway in ethanol's effects on growth, viability, and behavior, 2) to investigate the role of insulin signaling in the development of tolerance in ethanol-reared flies, and 3) to identiy ethanol's molecular targets through microarray analysis.

One of the goals of neuroscience is to understand how genes direct the development of the nervous system, and how sensory input from the environment interacts with the nervous system to result in behavior. Male fruit flies of the genus Drosophila carry out a complex and stereotyped courtship ritual that provides an excellent model in which to study these processes. The correct performance of this ritual is critical for success in mating and reproduction. Mutations affecting the courtship ritual typically result in slow courtship, or failure to court entirely. However, we have identified a gene (Tre1) that, when mutated, causes unusually rapid performance of the ritual. This result is unique, and suggests a previously unknown function in mating behavior: delay of courtship. This research project aims to answer two questions: first, why would a gene exist whose function appears to be to reduce the speed of mating, which would seem to put males at a disadvantage relative to males that mate more quickly? Second, in what cells does the Tre1 gene function, and what are these cells doing during courtship behavior? In addition, to increase the exposure of women and underrepresented minority students to basic scientific research, an integral component of this project is to establish a summer mentorship program that brings high school biology teachers from local minority-serving high schools, together with their students, to the lab to design and implement behavioral genetics experiments for their classrooms.

Drosophila males engage in a stereotyped courtship ritual in order to gain the favor of females. Previous research has shown that the typical result of failure to perform any of the steps correctly and in the correct order results in greatly reduced opportunities to mate. In addition, the behavioral sex determination gene fruitless (fru) has been shown to be necessary and sufficient to direct all steps of courtship behavior. Loss of the male-specific fru transcripts (fruM) typically leads to increased latency to court, inappropriate mate choice, or failure to court at all. We identified a GAL4-transgene that is inserted into the coding sequence of the Tre1 GPCR gene (Tre1-GAL4). When this GAL4 line is used to drive expression of either an RNAi targeting fruM or the feminizing transgene UAS-traF, it results in male flies that initiate courtship and achieve copulation much more quickly than control animals. This phenotype is recapitulated in males mutant for Tre1, which indicates that the Tre1 GPCR is required for normal courtship behavior. The expression pattern of Tre1-GAL4 is limited, with expression in regions consistent with olfactory reception and processing in both the peripheral and central nervous system. The activities described in this proposal identify positively the Tre1-GAL4 cells, investigate the function of those cells in courtship initiation, test the hypothesis that the Tre1-GAL4 cells are involved in mate choice, and further investigate the role of Tre1 itself in courtship initiation.

Project Summary: Developmental ethanol exposure causes a variety of deleterious phenotypes in taxa from insects to humans, including growth deficiency, developmental mortality, metabolic changes, intellectual disabilities, and behavior problems. In humans, these symptoms are collectively described as fetal alcohol spectrum disorder (FASD). Though epidemiological evidence suggests a minimum of 80,000 new cases of FASD every year in the United States alone, there is currently no approved biological treatment for FASD. Ethanol exposure is especially damaging to the developing nervous system and this has long-term consequences on adult behavior. The toxicity of developmental ethanol exposure has been attributed to numerous mechanisms, including ethanol metabolism and related oxidative stress, neuronal cell loss, and inhibition of growth factors and/or their signal transduction pathways. In particular, insulin and insulin-like growth factor (IGF) signaling is a universal target of developmental ethanol exposure. In mammals, the resulting insulin resistance leads to sensitivity to metabolic syndrome, which can be exacerbated by a high-fat diet. The goal of our research is to use the genetically amenable model organism Drosophila melanogaster to identify the molecular targets of developmental ethanol exposure, to understand how disruption of those targets leads to the deleterious phenotypes associated with developmental ethanol, and to test interventions that may one day lead to treatments for FASD in humans. Drosophila melanogaster, the common fruit fly, has been used extensively in biological research, particularly in genetics and development. Drosophila are particularly amenable to sophisticated genetic analyses, including genomic approaches, reverse and molecular genetics, and traditional forward genetic screens. Moreover, over a century of research has led to an extensive collection of genomic, molecular, genetic, and pharmacological tools, making Drosophila tremendously powerful in the elucidation of gene function. We have established and developed a genetic model of FASD in flies, and have used this model to show that insulin signaling is disrupted by developmental ethanol exposure, and that flies exposed to ethanol during development have phenotypes consistent with metabolic syndrome. We also have the first evidence that dietary changes may ameliorate the developmental effects of ethanol on metabolism. Finally, we discovered that developmental exposure to alcohol causes feeding deficits similar to those seen in mammals exposed to ethanol, and we have evidence implicating altered insulin signaling in this phenotype as well. Our research will further elucidate the role of insulin signaling in the development of FASD, by investigating the interaction between insulin signaling, diet, and predisposition to metabolic syndrome, as well as testing possible treatments for DAE-induced metabolic syndrome. In addition, we propose to further elucidate the molecular and neuronal signaling pathways that lead to changes in feeding behavior triggered by DAE. Our specific aims are: 1) to determine how diet and insulin signaling interact to mediate the toxicity of developmental ethanol exposure, and 2) to investigate the role of insulin signaling in the abnormal feeding behaviors that result from DAE.