Angel de Blas - US grants

DESCRIPTION: (Applicant's abstract) This research is aimed at understanding the regulation of the GABAa receptor (GABAaR) expression in the hippocampus and cerebellum of the rat brain associated to normal aging. Approximately 30-40% of the brain synapses are GABAergic. Nevertheless, the role of GABA and GABAaR in aging has seldom been studied in spite of suggestive data indicating the existence of changes in GABA and GABAaR during normal aging. One of the main reasons for the scarcity of studies in this area has been the absence of appropriate tools other than specific receptor radioligands. The recent development in the applicants laboratory of monoclonal and polyclonal antibodies and cDNA probes for the different receptor subunits will allow them to approach the role of GABAaR in aging in a very effective way. The applicants have already provided evidence and studied the aging-related changes in some GABAaR subunits and GAD expression in some areas of the brain. The techniques involved are I) quantitative in situ, northern and dot blot hybridization for studying the mRNA expression of receptor subunits; II) quantitative immunocytochemistry, immunoblotting and immunoprecipitation for studying the protein expression; III) quantitative radioligand autoradiography for studying the ligand binding specificities of the receptor since it is expected that changes in receptor subunit composition leads to changes in binding affinities for some ligands. In addition, deafferentation models of the hippocampus and cerebellum will be used to test the hypothesis that some of the aging-related changes in GABAaR subunit expression are due to changes in neuronal connectivity. The possible involvement of neurotrophins and their receptors in the regulation of GABAaR subunit expression will also be investigated. These studies are directly relevant to aging in several ways: I) Much of the physical and mental deterioration associated with aging is due to impairments in brain physiology. Results obtained in our laboratory as well as in others indicate that the GABAaR change during aging. II) Memory, frequently is impaired during aging. Published results have shown the importance of the hippocampal GABAaR for the consolidation of memory. III) Benzodiazepines (ie. Librium and Valium) exert their action by binding to the GABAaR. Sleep disorders frequently accompany aging. Benzodiazepines are widely used to treat sleep disorders. IV) The GABAaR are also involved in the control of blood pressure and in areas of the brain involved in sensory-motor coordination, hearing and vision. In addition, the GABAaR are prominently involved in the physiology of the retina. Vision, hearing and motor coordination are frequently impaired during aging. V) Understanding of the aging-related changes in GABAaR subunit composition and ligand binding will help to develop drugs that would recognize specific types of GABAaR involved in an aging-related impaired function without affecting other GABAaR involved in other brain functions, thus eliminating most of the drug side-effects.

DESCRIPTION: (Applicant's Abstract) It has been estimated that 40 percent of the brain synapses are GABAergic. However, little is known about the mechanisms by which GABAA receptors (GABAAR) are concentrated at inhibitory GABAergic Gray's type 2 synapses. The long-term objective of this proposal is to reveal the molecular components of the GABAergic Gray's type 2 postsynaptic density and to understand the molecular mechanisms involved in clustering and anchoring GABAAR at the postsynaptic membrane. The specific aims of this proposal are directed towards revealing the cytoplasmic proteins that directly bind to GABAAR and to test whether these proteins are responsible for the clustering and/or localization of the GABAAR at synapses and/or other membrane domains. The aims will test the hypotheses that 1) a novel GABAAR protein is involved in GABAAR clustering and/or anchoring at GABAergic synapses; 2) in neurons, the interaction of the beta3 GABAAR subunit with a guanine nucleotide exchange protein is necessary for GABAAR transport to the cell surface; and 3) the novel proteins that interact with various GABAAR subunits in the yeast two-hybrid screens also interact with the GABAAR in neurons and are involved in receptor clustering or anchoring at synapses. Yeast two-hybrid assays are being used in our laboratory to identify GABAAR binding proteins. The functionality of the interaction between GABAAR and binding proteins will be tested by various in vitro biochemical assays as well as by in vivo (in culture) assays including primary hippocampal neuronal cultures and cotransfected host cells. The synaptic colocalization of the GABAA receptors and interacting proteins will be studied by both light microscopic immunofluorescence and electron microscopic immunogold immunocytochemistry using specific antibodies to GABAAR subunits and interacting protein. The GABAAR and interacting protein amino acid sequences involved in protein-protein interactions will be identified. These studies will help to better understand the molecular components and interactions involved in the structure, function and modulation of the inhibitory GABAergic synaptic transmission as well as the mechanisms involved in neuronal connectivity and synaptic plasticity. It is predicted that the inappropriate synaptic and extrasynaptic localization of the GABAAR, affects GABAergic synaptic function and brain development, leading to neurological and mental disorders.

DESCRIPTION: (Applicant's Abstract) It has been estimated that 40 percent of the brain synapses are GABAergic. Nevertheless, little is known about the the possible relationship between the GABAA receptor subunit composition and the synaptic and extrasynaptic localization of the receptor. The long-term objective of this proposal is to understand the molecular mechanisms involved in clustering and anchoring GABAA receptors at the postsynaptic membrane. The specific aims of this proposal are: 1) To test the hypothesis that the presence of gamma (gamma1, gamma2s, gamma2L or gamma3) subunits in the GABAAR pentamer in association with gephyrin and other synaptic proteins is necessary to cluster and position the GABAAR at GABAergic synapses; 2) To test the hypothesis that the alpha4 subunit in the hippocampus and thalamus associates with either the delta subunit or with a gamma subunit. The former GABAAR are extrasynaptic, the latter are synaptically localized; 3) To determine the cellular, subcellular, synaptic and extrasynaptic localization of beta1, beta2 and beta3, subunits in the rat brain in order to generate hypotheses about the possible relationships between beta subunit isoform and receptor localization and to test the hypothesis that the beta3 subunit is preferentially localized at synapses. Already available subunit-specific GABAA receptor antibodies in combination with immunocytochemistry at the light microscopy and EM levels will be used for revealing the cellular and subcellular localization of various GABAAR subunits at high resolution. We will do immunofluorescence in low density hippocampal cultures and immunogold at the EM level in intact rat hippocampus and thalamus and in mouse knockout mutants. Antisense treatment and transfections of primary hippocampal neuronal cultures will also be used to probe the mechanisms of subunit targeting to synapses. These studies will be relevant for understanding the mechanisms involved in the formation, maintenance and plasticity of the inhibitory GABAergic synapses in the brain. It is predicted that the inappropriate synaptic and extrasynaptic localization of the GABAA receptors, affects GABAergic synaptic function and brain development, leading to neurological and mental disorders.

DESCRIPTION (provided by applicant): There is limited knowledge about the relationship between the GABAA receptor subunit composition and the synaptic, extrasynaptic and other subcellular localization of this receptor. This is in spite of the importance of the GABAA receptor in brain function. It has been estimated that 40% of the brain synapses are GABAergic. The long-term objective of this proposal is to understand the molecular mechanisms involved in clustering and anchoring GABAA receptors at the postsynaptic membrane. The aims of this proposal are: 1) To identify the amino acids of various subunits involved in the synaptic clustering of the GABAA receptors; 2) To study the synaptic and extrasynaptic localization of the gamma 3 and alpha 5 subunits and 3) To biochemically separate and characterize synaptic and non-synaptic GABAA receptors from various subcellular fractions isolated from the brain. Already available subunit-specific GABAA receptor antibodies in combination with immunocytochemistry at the light microscopy and electron microscopy levels will be used for revealing the cellular, subcellular and synaptic localization of various GABAA receptors in the rat brain and in hippocampal cultures. The same antibodies will be used for the biochemical characterization of the GABAA receptors in the subcellular fractions isolated from the brain. Techniques such as siRNA treatment of cultured hippocampal neurons and the expression of mutated GABAA receptor subunits in these cultures and in the brain will also be used aiming to elucidate the mechanisms involved in the synaptic clustering of GABAA receptors. These studies are relevant for understanding the mechanisms involved in the formation, maintenance and plasticity of the inhibitory GABAergic synapses in the brain. It is predicted that the inappropriate synaptic and extrasynaptic localization of GABAA receptors affects GABAergic synaptic function and brain development, leading to neurological and mental disorders. These studies will also be relevant for designing GABAergic therapeutic drugs with higher clinical specificity and fewer side effects.

There is limited knowledge about the mechanisms by which GABA-A receptors are concentrated at inhibitory GABAergic Gray's type-2 synapses. The GABA-A receptors play a major role in brain function. It has been estimated that 40% of the brain synapses are GABAergic. The long-term objective of this proposal is to identify the proteins of the type-2 GABAergic synapses involved in the postsynaptic clustering and anchoring of GABA-A receptors. The aims of this proposal are:1) To demonstrate that GRIP proteins are involved in the postsynaptic clustering of gephyrin and GABA-A receptors;2) To study the the relationship between the synaptic clustering of a protocadherin, whose intracellular domain interacts with the large intracellular loop of the gamma 2 subunit of the GABA-A receptor, and the synaptic clustering of GABA-A receptors and 3) To test the hypothesis that septins 6 and 7, which are enriched in type-2 postsynaptic densities in the rat brain, are involved in GABA-A receptor clustering and/or exocytosis. Light microscopy and electron microscopy immunocytochemistry with specific antibodies will be used for revealing the synaptic co-localization and clustering of GABA-A receptors and synaptic proteins in the intact brain and in hippocampal cultures. Identification of synaptic proteins will be done by mass spectrometry. Techniques such as siRNA treatment of cultured hippocampal neurons and the expression of tagged postsynaptic proteins in these cultures and in the intact brain will also be used to elucidate the synaptic targeting and clustering of the synaptic proteins and GABA-A receptors. These studies are relevant for understanding the molecular components of GABAergic synapses, their synaptic targeting and postsynaptic clustering as well as the mechanisms involved in neuronal connectivity and synaptic plasticity. It is predicted that the inappropriate synaptic and extrasynaptic localization of GABA-A receptors affects GABAergic synaptic function and brain development, leading to epilepsy and other neurological and mental disorders.