Ulrike Heberlein - US grants

[unreadable] DESCRIPTION (provided by applicant): The goal of this proposal is to study the mechanisms by which insulin-like peptides and the insulin receptor pathway regulate ethanol-induced behaviors in Drosophila and in mice This idea emerged from the convergence of two independent lines of investigation. First, previous genetic screens for Drosophila mutants with altered ethanol sensitivity identified mutations in several components of the insulin receptor signaling pathway. Second, neuroanatomical mapping experiments in Drosophila revealed that a group of insulin producing neurosecretory cells controls normal ethanol sensitivity. We therefore postulate that the insulin pathway, well known for its role in regulating metabolism, growth, and life span, is also involved in the regulation of ethanol-related behaviors. Here we propose to use genetic, and molecular approaches to define how, when, and where insulin functions in ethanol sensitivity in Drosophila and to begin the translation of this information to a mammalian system. First, we will study the role of insulin-producing cells in ethanol-related behaviors. Second, we will use a collection of existing mutants and transgenic fly strains, which are known to inhibit or activate various components of the insulin receptor pathway, to define additional signaling molecules and to establish their functional relationships. Third, we will use inducible gene expression strategies to determine if the insulin pathway functions during development or adulthood to regulate ethanol-induced behaviors. Fourth, we will study the functional relationship between insulin-like peptides and the neuropeptide encoded by the amnesiac gene, which was previously shown to regulate ethanol sensitivity. Finally, we will test if this signaling pathway plays a role ethanol-related behaviors in mice. [unreadable] [unreadable] The role of the insulin pathway in regulating growth, metabolism, and life span has been shown to be conserved from invertebrates to mammals. We predict that this functional conservation will hold for ethanol related phenomena. As drugs that target components of the insulin pathway are already available, information gained from studies in flies and mice could be readily transferred to people with alcohol abuse or alcoholism problems. [unreadable] [unreadable]

The objective of the proposed research is to understand, at the molecular level, the mechanisms underlying the early phases of retinal morphogenesis in Drosophila. This is a complex process by which a simple unpatterned epithelium begins to differentiate into a highly organized retina. The precision with which this developmental process is carried out is critical for achieving a fully functional visual system. Some of the molecules known to be involved in the process have homologues that are likely to execute similar functions during vertebrate retinal development. We will approach this objective by identifying novel genes, by means of a genetic screen, that carry out the various steps required for proper retinal morphogenesis. These genes should include those involved in the adoption and restriction of neural fate, in the appropriate patterning of photoreceptor precursors, and in the propagation of this pattern across the differentiating retinal epithelium. We will characterize these genes and study the phenotypic consequences of their loss and gain of function. This will be carried out by a combination of genetic, molecular, histochemical and, eventually, biochemical methods. This analysis should establish precisely what the roles of the genes are during eye development. Ultimately, these genes will provide useful tools for the process of integration, the goal of which is to establish how the various isolated components work in concert to give rise to the adult retina. Because of the emerging parallels between Drosophila and vertebrate eye development, we believe that the knowledge gained from this and similar studies will be applicable to the normal development of vertebrate retinae as well as to situations of genetic or environmental injury.

DESCRIPTION: (provided by the applicant) Nicotine addiction is a major health problem in the world, yet little is known about the molecular and cellular mechanisms that mediate its addicting effects. The goal of this project is to develop "Drosophila melanogaster" as an animal model to identify the genes and signaling pathways that regulate acute sensitivity to nicotine, the development of tolerance, and withdrawal. For this purpose we plan to carry out a genetic screen for mutants that show aberrant behaviors upon acute and chronic exposure to nicotine. The acute effects of nicotine will be measured with assays for locomotion, geotaxis, and postural nicotine. Tolerance will be quantified as the change in acute responses caused by pre-feeding nicotine for several days. Mutants that show altered responses that are not accounted for by changes in sensory systems will be characterized molecularly. We also propose to develop assays to quantify withdrawal from chronic nicotine exposure. These include assays for general hyperactivity and startle responses to chemosensory and mechanosensory stimuli. Flies are easy and inexpensive to rear, and nearly a century of extensive analysis has provided innumerable and sophisticated tools for genetic and molecular analysis. These attributes, together with the high degree of evolutionary conservation and the relevant neurochemical systems, allow us to carry out unbiased screens for novel molecules involved in nicotine responses, an approach that would be very expensive and laborious to implement in mammals. The genes identified in "Drosophila" should provide potential candidate genes and signaling pathways to be studied in rodent models and in human genetic studies.

DESCRIPTION (provided by applicant): In most model organisms in which it has been studied, low doses of ethanol have central nervous system- activating effects, whereas high doses are sedative. These acute effects of ethanol are commonly measured as changes in locomotor activity. Interestingly, a growing body of evidence suggests that the molecular, neurochemical, and neuroanatomical mechanisms that regulate ethanol's effect on locomotion are at least in part shared with those that underlie the drug's rewarding effects. For example, pharmacological and genetic manipulations have shown that midbrain dopaminergic pathways are involved in both the activating and rewarding effects of ethanol in rodent models. It is therefore likely that insights into ethanol's addicting effects can be obtained by studying the relatively simple effects of the drug on locomotor activity. The goal of this proposal is to study the genetic, molecular, and neural mechanisms that regulate ethanol- induced locomotor behaviors in Drosophila. Preliminary experiments have revealed that fruitflies show a biphasic locomotor response to ethanol similar to that observed in mammals, and that, as in rodents, dopaminergic systems play a role in ethanol's acute activating effects in flies. We propose to generate and characterize mutants with altered ethanol-induced locomotor activity. The genes disrupted by these mutations will be studied molecularly and their function will be analyzed in vivo upon establishment of transgenic flies. The brain regions that mediate ethanol's effect on locomotion will be mapped by selectively inactivating specific brain regions by means of targeted expression of a tetanus toxin-encoding transgene. The neurochemical profiles of these brain regions will be ascertained by determining the expression of neurotransmitters, the enzymes involved in their synthesis, and/or the receptor systems upon which they act. Together, these approaches will help us understand the basic molecular pathways and neural circuits that regulate ethanol-induced locomotor behaviors in Drosophila. Because the mechanisms of ethanol's action share common features in organisms as different as flies and mice, we predict that knowledge gained from Drosophila will provide novel and general insights into the processes by which ethanol acts in the nervous system to regulate behavior.

[unreadable] Description (provided by applicant): The goal of this proposal is to study the mechanisms by which the LIM-domain only (LMO) proteins affect psychostimulant-induced behaviors in Drosophila and in mice. In a genetic screen for Drosophila mutants with altered responsiveness to acute nicotine and cocaine exposure, multiple alleles of the Lmo gene were isolated. LMOs are highly-conserved proteins that regulate the activity of LIM-homeodomain (LIM-HD) factors. These, in turn, regulate multiple aspects of the final differentiated state of neurons, such as their neurochemical identity and receptor expression profile. LMOs are thus ideally suited to modulate in a subtle manner the sensitivity of the nervous system to drugs of abuse. We propose to use genetic, molecular, genomic, and neuroanatomical approaches to determine when, where and how Lmo functions in the Drosophila brain to regulate psychostimulant responsiveness. In addition, we will begin to translate this information to a mammalian system by studying the role of LMOs in psychostimulant-induced behaviors in mice. The Specific Aims of the proposal are as follows. First, we will use inducible gene expression strategies to determine whether LMO acts in the developing and/or adult nervous system to regulate drug behaviors in Drosophila. Second, we will use a series of genetic experiments in Drosophila to define the identity of the functional LIM-HD partner(s) for LMO in the context of cocaine and nicotine sensitivity. Third, we will use transcriptional profiling to define the genes whose expression is altered by loss or gain of Lmo function in Drosophila. Fourth, we will test the role of mouse Lmo homologs in several behavioral paradigms that measure the acute stimulant and rewarding properties of cocaine. LMOS and LMO4 were chosen for this analysis as they are expressed in brain regions implicated in the reinforcing properties of abused drugs. [unreadable] [unreadable] Together, these experiments will provide novel insights into the mechanisms by which psychostimulant sensitivity is controlled in the nervous system. LMOs have been shown to function as highly dynamic transcriptional modulators and are thus well-suited to regulate adaptive and potentially long-lasting changes in nervous system physiology. Exposure to drugs of abuse leads to long-term changes in the brain that are believed to cause the addicted state. LMOs are attractive candidate molecules to mediate these changes. The relevance of this project is that it will help us understand how abused drugs, such as cocaine, cause long-lasting changes in the brain. By interfering with these changes, or reversing them, we should be able to develop therapies to help treat drug addicts. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Alcohol is one of the most popularly consumed and abused drugs in the world. The pleasurable and disinhibiting effects of alcohol consumption have been enjoyed by humankind for thousands of years. For some, however, alcohol consumption leads to alcohol addiction, a devastating illness with enormous medical and societal costs. In humans, long-term alcohol use leads to the development of tolerance, most simply defined as an acquired resistance to the inebriating effects of ethanol. Alcoholics can appear sober at blood alcohol levels that would be severely intoxicating or even fatal to naive individuals. The overall goal of this proposal is to decipher the relationship between the molecular mechanisms contributing to ethanol tolerance and those that mediate the addicting effects of the drug. Drosophila melanogaster, with its accessibility to genetic, behavioral and molecular analyses, has been developed as a powerful model system to study the mechanisms underlying ethanol intoxication and tolerance development. In this proposal, we plan to study a collection of existing mutants with defects in ethanol tolerance in a series of newly-developed assays that quantify ethanol consumption, preference, conditioned preference, and relapse in Drosophila. These studies will provide new insights into the relationship between the molecular mechanisms of ethanol tolerance and those underlying the rewarding and addicting properties of ethanol. In addition, these genes and their products will be attractive candidate targets for therapeutic intervention. PUBLIC HEALTH RELEVANCE: Low doses of ethanol in naive individuals lead to intoxication, with pleasurable effects such as euphoria and loss of social inhibitions, as well as aversive effects such as loss of motor coordination, dehydration, and sedation. However, chronic exposure to high ethanol doses is toxic, leading to severe and irreversible damage to the brain, liver, and kidneys. Alcohol abuse is facilitated by the development of tolerance, most simply defined as an acquired resistance to the effects of the drug. In humans, tolerance is thought to develop rapidly to the aversive effects of ethanol, and to a lesser extent to its pleasurable properties. This initial imbalance between aversive and enjoyable effects has been suggested to encourage greater intake, which over time leads to the development of physical dependence and possibly addiction. In this proposal we plan to study the relationship between ethanol tolerance and ethanol addiction, using the powerful molecular genetics offered by the model organism Drosophila melanogaster. These studies will provide new insights into the relationship between the genes involved in ethanol tolerance and those mediating the rewarding and addicting properties of ethanol. These genes and their products will be attractive candidate targets for therapeutic intervention.