Bruce A. Bamber - US grants

DESCRIPTION (provided by applicant): The objective of this developmental/exploratory R21 proposal is to develop a new direction for studying GABAA receptor trafficking and regulation at synapses. A genetic model organism approach will be taken, using the nematode Caenorhabditis elegans. GABAA receptors are the major inhibitory neurotransmitter receptors in the brain, and play important roles in most brain functions. The abundance of GABAA receptors at synapses is an important factor in normal nervous system function, and in the progression of nervous system disease, particularly anxiety disorders. Studies in mammalian systems suggest that receptor abundance is regulated by controlling receptor biosynthesis, trafficking to synapses, and degradation. However, results to date have been largely descriptive. We lack an understanding of how these processes occur, and how they may be regulated. Progress has been slowed by the difficulty of perturbing mammalian gene expression, the anatomical complexity of the mammalian nervous system, and the structural heterogeneity of mammalian GABAA receptors. One solution is to take a genetic approach in a simple model organism. C. elegans is well suited for this purpose: GABAA receptors, and their regulation at synapses, are conserved in C. elegans; genetic manipulation of C. elegans is rapid and efficient; the C. elegans nervous system is relatively simple; and the C. elegans GABAA receptor is uniform and well characterized. The specific aims of this proposal are two-fold: First, the points along the biosynthetic, trafficking, and degradation pathways that control GABAA receptor abundance at synapses will be determined. To achieve this aim, these points will be perturbed genetically, and the effects on the regulation of receptor abundance will be determined. Measurements of receptor function (using electrophysiology), receptor subcellular localization, and receptor mRNA and protein levels will be performed. Second, the genes that are important at each of these potential regulatory points will be determined. This aim will be achieved by forward genetic screening using a functional GFP-tagged receptor. These initial data will form the basis of a substantial future research program to understand how GABAA receptor abundance at synapses is regulated. Because cellular processes are generally well-conserved between C. elegans and humans, insights from these studies could lead to improved treatments for anxiety disorders based on modulating GABAA receptor abundance at synapses.

[unreadable] DESCRIPTION (provided by applicant): Epilepsy is a debilitating disease that affects 1-2% of the population. Anticonvulsant drug therapy is the treatment of choice, however about 30% of epilepsies are intractable to drug therapy, and necessitate surgical removal of brain tissue. In addition, anticonvulsant drugs reduce neuronal excitability non-specifically throughout the brain, leading to sedation and somnolence as side effects. Thus, there is an ongoing need to develop new anti-epileptic drugs. The overall goals of the proposed research are to help develop new anti-epileptic drug therapies by investigating the mechanisms of a new class of anticonvulsant drugs, the neurosteroids. Neurosteroids are synthesized in the brain, and regulate neuronal excitability by modulating GABAA receptors (the major inhibitory neurotransmitter receptors in the brain). Synthetic neurosteroids have proven to be effective anti-epileptic drugs in clinical trials, even against otherwise drug resistant epilepsies. Moreover, neurosteroids can potentially be engineered for greater specificity, to reduce side-effects. Two approaches will be taken to better understand neurosteroid modulation of GABAA receptors. First, to gain fundamental insights into neurosteroid mechanisms, a simple, well-characterized steroid-receptor interaction will be studied: The inhibition of the C. elegans UNC-49 receptor by pregnenolone sulfate (PS). Rapid ligand exchange methods will be used to characterize the mechanism of PS modulation of UNC-49, and to determine how the key residues act as effectors of PS modulation. Results of this study will be applicable to understanding neurosteroid modulation in humans because UNC-49 is a highly conserved GABAA receptor homologue. Second, the unique pharmacological properties of UNC-49 will be exploited in domain swap experiments to directly identify residues important for neurosteroid modulation in human GABAA receptors. The results of both approaches will increase understanding of how endogenous neurosteroids regulate seizure susceptibility, and will aid the design of new anti-epileptic drugs. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): GABA is the principal inhibitory neurotransmitter receptor in the human brain, functioning primarily through GABAA receptors. Normal brain function is critically dependent on the strength of GABA synapses, which in turn depends on the abundance postsynaptic GABAA receptors. Abnormalities in GABAA receptor expression are observed in schizophrenia, alcoholism, anxiety disorders and epilepsy, and in some cases are known to play a causal role in disease progression. Drugs that potentiate GABAA receptor function are important tools for physicians to treat anxiety disorders, epilepsy, and insomnia. Therefore, it is important to understand the mechanisms that regulate the abundance and function of GABAA receptors at synapses. This proposal is directed toward understanding how postsynaptic cells regulate GABAA receptor abundance, using the simple model organism Caenorhabditis elegans. C. elegans has a small nervous system, and can be easily studied by physiological, imaging, and genetic techniques. Significantly, considerable evolutionary conservation has been uncovered at the molecular level between GABA neurotransmission in C. elegans and humans, so what we learn in worms will be medically significant. GABA receptors in mammals are regulated homeostatically in response to agonist exposure and alteration of cellular and network activity. We observe comparable regulation of GABA receptors in C. elegans. Using the power of C. elegans genetic and molecular analysis, we will probe the mechanistic basis of GABA receptor homeostatic regulation, with the eventual goal of understanding the relevant signal transduction pathways. This information will be directly applicable to understanding GABA receptor regulation in both normal and abnormal brain function. PUBLIC HEALTH RELEVANCE: Normal brain function is critically dependent on the strength of GABA synapses. The primary determinants of GABA synapse strength are the quantity of GABA released from presynaptic nerve terminals, and the abundance postsynaptic GABAA receptors. Abnormalities in GABAA receptor expression are observed in schizophrenia, alcoholism, anxiety disorders and epilepsy, and in some cases are known to play a causal role in disease progression. This proposal is directed toward understanding the endogenous mechanisms that regulate the strength of GABA synapses, with particular emphasis on mechanisms that regulate GABAA receptor abundance.

DESCRIPTION (provided by applicant): Nematode infections are a major cause of human morbidity and contribute significantly to a loss of Disability Adjusted Life Years. More importantly, in many cases, such as filarial infection, effective chemotherapy is still not availabe. Perhaps less well appreciated, but equally important for human health, is the devastating economic impact of parasitic nematodes on livestock and plants, and new anthelmintics and drug targets are both desperately needed in all settings. Most anthelmintics in use against nematode infections act as agonists at key receptors and cause paralysis by interfering with muscle contraction and/or locomotion. The overall objective of this renewal application of our previously funded grant 'Locomotion in Parasitic Nematodes' (AI072644) is to promote the development of new anthelmintics that target locomotion to cause paralysis. In the previous funding period we characterized key monoamine receptors regulating locomotion using an innovative 'dual systems' approach that exploited the experimental advantages of the C. elegans and Ascaris suum models. Highlighting the utility of this approach, C. elegans molecular genetics was instrumental in isolating key receptor genes; bioinformatics approaches then identified corresponding parasitic nematode cDNAs, and ultimately, four key monoamine receptors were identified as promising possible anthelmintic targets. In the present study we will develop innovative anthelmintic drug screening strategies based on these receptors by characterizing their agonist sensitivities in heterologous cells, and expressing them in C. elegans, to create 'chimeric' nematodes to confirm orthology and allow agonist screening under physiological conditions. We will establish the sites of action and physiological roles of these receptors in the locomotory circuitry of parasitic nematodes by direct functional localization and electrophysiological approaches. We will also continue the dual-systems approach, using the well-developed C. elegans 'toolkit' to investigate signaling pathways downstream of these receptors, and define their precise roles in 'central' circuits that make locomotory decisions. These studies will not only identify definitively key monoamine receptors regulating locomotion in parasitic nematodes, but because of the enormous diversity among nematodes, they will also highlight the potential differences between these two important model systems. Locomotion is critical to the survival of parasitic nematodes, and drugs that inhibit locomotion can successfully clear parasitic nematode infection. These studies will identify a wealth of potential novel molecular targets for drug discovery.