Mechanisms that regulate the trafficking and clustering of postsynaptic GABA-A receptors (supported by NIMH, R01 MH62391)

The long-term goals of the proposed research are to unravel mechanisms that regulate trafficking and postsynaptic clustering of gamma-aminobutyric acid type A (GABA-A) receptors. These receptors are hetero-pentameric chloride channels and they mediate most inhibitory neurotransmission in the brain. GABA-A receptor subtypes distinguished by their subunit composition are differentially expressed during development and in different regions of the mature brain. Most GABA-A receptor subtypes are clustered at postsynaptic sites but the subunit requirements and other factors involved in subcellular localization are ill-defined. Differential synaptic localization of GABA-A receptors is implicated in regulation of synaptic specificity and efficacy of GABAergic transmission and pathological changes in receptor localization can be causal for neurological and mental disorders such as epilepsy and anxiety. In particular, the factors and signaling pathways that determine receptor clustering and postsynaptic localization are largely unknown and shall be identified as part of this project. We hypothesize that synaptic localization is mediated by specific interactions of GABA-A receptor with diverse postsynaptic factors that are involved in trafficking of GABA-A receptors or act as diffusion traps. Initial studies in our lab have identified the gamma2 subunit as an important determinant of postsynaptic localization of GABA-A receptors (Essrich et al. 1998, Schweizer et al 2003). More recently we have mapped the gamma2 subunit domains that contribute to postsynaptic accumulation of GABA-A receptors, Unexpectedly, we found that postsynaptic localization is largely independent of cytoplasmic receptor domains but mediated by the fourth transmembrane domain (TM4) of the gamma2 subunit. However, proper function of postsynaptic GABA-A receptors requires both the TM4 and the major cytoplasmic loop domain of the gamma2 subunit (Alldred et al. 2005).

In another approach we are screening cDNA libraries for GABA-A receptor interacting proteins that might contribute to trafficking of postsynaptic GABA-A receptors. Using the gamma2 subunit cytoplasmic loop domain as a bait in a yeast two-hybrid screen (SOS recruitment system) we have identified GODZ as a first member of mammalian palmitoyltransferases (Keller et al 2004). GODZ palmitoylates several cysteines in the cytoplasmic loop domain of the gamma2 subunit, a mechanism that is implicated in trafficking of gamma subunit-containing GABA-A receptors to the plasma membrane and to postsynaptic sites. Indeed palmitoylation is a reversible posttranslational modification of diverse cytoplasmic proteins especially in neurons that might contribute to dynamic modulation of the clustering and function of postsynaptic GABA-A receptors.

The specific behavioral and cognitive deficits of gamma2 subunit heterozygous (gamma2 0/+) mice, together with established knowledge on the neural circuitry of conditioned fear and the regional distribution of the GABA-A receptor deficit in gamma2 0/+ mice allow predictions as to which brain regions mediate trait anxiety.

We hypothesize that GABAergic deficits in the cerebral cortex and/or the hippocampus underlie behavioral responses and cognitive deficits indicative of trait anxiety. Enhanced contextual processing in these brain regions is believed to be causal for anxiety disorders that are associated with trait anxiety. In order to test this hypothesis, we use the gamma2 subunit gene as a molecular tag to directly localize the neural circuits and brain regions that mediate trait anxiety. A mouse line has been generated that allows conditional and spatio-temporally restricted inactivation of the gamma2 subunit gene by means of the Cre/loxP system. Upon Cre-induced inactivation of the gamma2 subunit gene, GABA-A receptor function will be impaired. Region-specific Cre-transgenes and stereotaxic application of an adeno-associated virus (AAV-CRE/GFP) is being used to limit and map the deficit to small brain regions. It is predicted that a regionally confined deficit in some brain regions but not in others results in trait anxiety-like behavior as described for globally mutated gamma2 0/+ mice. Cre-transgenic mouse lines that selectively target the relevant brain areas (cerebral cortex, hippocampus) are available to directly test this hypothesis. Other brain regions known to be essential for fear conditioning such as the amygdala and the septum will be tested using stereotaxic application of AAV-CRE/GFP to induce the GABAergic deficit.

Research in our lab revolves around understanding the structure of GABAergic inhibitory synapses including GABA-A receptors, receptor-associated proteins, their posttranslational modifications and how this relates to dynamic functional modulation of synapses both under physiological conditions and in neurological and mental disorders.

GABA (gamma-aminobutyric acid) is the major inhibitory neurotransmitter in the brain and mediates the vast majority of neural inhibition by activation of so-called GABA-A receptors. Structurally, GABA-A receptors are heteropentameric chloride channels that include a number of different binding sites for clinically important drugs, most notably the benzodiazepines (BZs) and diverse anesthetic agents. By modulating GABA-A receptor function, ligands of the BZ site can enhance or inhibit a wide variety of CNS states, including vigilance, anxiety, epileptic activity, muscle tension and memory. Thus, GABA-A receptors serve as important targets for therapeutic modulation of various CNS functions and their proper regulation is critical for maintaining normal brain function and mental health. Of particular interest is the role of GABA-A receptor deficits in emotional disorders (anxiety disorders, depression), epilepsy and schizophrenia.

The Long-Term Objectives of our research have in common that they deal with GABA-A receptors: · First we would like to understand the molecular and cellular mechanisms controlling the concentration of GABA-A receptors at different types of inhibitory synapses and how these mechanisms relate to functional modulation of inhibitory synapses in vitro and in vivo. · Second, we are taking advantage of sophisticated animal models that exhibit spatially and temporally controlled GABA-A receptor deficits to analyze the role of GABAergic transmission, and downstream signal transduction pathways, that determine normal emotionality during development and in the adult brain.