- Drug Discovery
- The NMDA Receptor
- Gene Regulation
- Gene Therapy
- The GABAA Receptor
- The GABAB Receptor
Nervous tissue synthesizes steroids (neurosteroids) from cholesterol independently of the endocrine system. Whereas steroids are known to regulate diverse cellular functions via interaction with cytosolic steroid hormone receptors and their genomic response elements, certain neurosteroids have been found to exert non-genomic modulatory effects through direct interaction with neurotransmitter receptors and ion channels. Our laboratory discovered that pregnenolone sulfate (PS), the most abundant neurosteroid in the mammalian nervous system, regulates the activity of the gamma-amino-butyric acid type A receptor (GABAA R) and ionotropic glutamate receptors (iGluRs), including the N-methyl-D-aspartate receptor (NMDAR), and may act as a neurotransmitter or neuromodulator.
Ongoing research focuses on the mechanism of action and discovery of neuromodulators as therapeutic agents and on the structure, function, and cellular dynamics of ion channels and amino acid receptors in the brain and spinal cord. Abnormal activation of amino acid receptors has been implicated in the etiology of neuropsychiatric disorders such as substance abuse, anxiety, depression, and schizophrenia as well as of seizure disorders. Amino acid receptor function can be controlled by direct modulation of receptor function on a time scale ranging from milliseconds to minutes, and by slower long-term regulation of receptor gene expression on a time scale of hours to days.
Current studies address the pharmacology of neurotransmitters and neuromodulators, including benzodiazepines, barbiturates, and steroids, and mechanisms of modulation and regulation of neurotransmitter receptor function, including up-regulation, down-regulation, desensitization, and tolerance. Computational and electrophysiological methods are used to evaluate thermodynamically plausible models for receptor function.
We have isolated segments of DNA from the human genome that contain the genetic blueprint for the production of GABA receptors. By determining the sequences for the “promoter” regions of the gene that control its expression, we are identifying receptor-specific transcription factors, such as an ethanol responsive transcriptional element or ” ERTE”. This will provide a basis for designing new classes of therapeutic agents that act by regulating the expression of neurotransmitter receptors and their transcription factors.
A high-throughput electrophysiology (HTEP) has been developed which allows for rapid electrophysiological screening of compound libraries for activity on receptors of defined subunit composition, to facilitate the discovery of more selective agonists, antagonists, and modulators
The NMDA Receptor
The NMDA receptor is a member of the ligand-gated ionotropic glutamate receptor family that mediates most of the excitatory synaptic transmission in the mammalian central nervous system. The slower phase of the post-synaptic response elicited by glutamate at the majority of excitatory central synapses has been attributed to activation of the receptor. NMDA receptors are expressed in neurons throughout the nervous system and are essential for neuronal development and synaptic plasticity. However, over-excitation of NMDA receptors is associated with certain acute and chronic neurological disorders, including stroke, neuropathic pain, and neurodegenerative diseases.
A characteristic of the NMDA receptor that distinguishes it from other ion channels is that the binding of multiple agonists (glutamate and glycine) and the relief of the Mg2+ block by membrane depolarization are prerequisites for ion transport. Therefore, the NMDA receptor is often referred to as a molecular coincidence detector that can integrate information from pre- and post-synaptic events. Ca2+ entering through NMDA receptors can trigger cascades of signaling pathways that can generate persistent changes in synaptic efficacy via both pre- and post-synaptic mechanisms. Long-term changes in synaptic responses, either potentiation or depression (LTP or LTD), have been a dominant cellular model for learning and memory.
Our chief interests surround a desire to understand the dynamic regulation of gene expression in the nervous system. The identification of gene families with multiple genes that code for related yet distinct receptor isoforms has added a remarkable increase to the level of specificity and complexity that governs the transcriptional regulation of neurotransmitter receptors in the CNS. For instance, the diverse set of nineteen genes coding for GABAA receptor subunits constitutes a gene family that displays an unusual degree of differential developmental and cell-specific expression. This expression leads to pharmacological diversity that enables individual neurons to respond dynamically to alterations in membrane excitability.
As a first step in the process of investigating transcriptional control in the CNS, we have employed a variety of techniques, including yeast hybrid assays, chromatin immunoprecipitation, and transfection into primary neuronal cultures, to identify key regulatory elements and transcription factors that specifically enhance or repress expression of GABAA and NMDA receptor subunits.
We have also begun to use adeno-associated vectors (AAV), engineered with subunit specific promoters, in vivo to test several models for the etiology of temporal lobe epilepsy.
The GABAA Receptor
GABAA receptors are primarily responsible for fast inhibitory synaptic transmission in the brain. Drugs that enhance GABA mediated neurotransmission are widely used clinically as anticonvulsants, antianxiety agents, and sedatives. A diverse set of nineteen genes coding for GABAA receptor subunits constitutes a gene family that displays an unusual degree of differential developmental and cell-specific expression. Combinatorial assembly of GABAA receptor subunits into multiple receptors subtypes offers the potential for development of therapeutic agents acting upon GABAA receptors with improved selectivity.
The GABAB Receptor
The slow inhibitory action of GABA is mediated by metabotropic GABAB receptors. Disruption of GABAB receptor-mediated synaptic pathways has been implicated in many diseases, including neuropathic pain, spasticity, drug addiction, schizophrenia, and epilepsy. Although the GABAB receptor agonist baclofen has been used in the clinic for over thirty years, the molecular nature of the GABAB receptor was only recently elucidated to be a heterodimer of two distinct subunits GABABR1 and GABABR2. Our laboratory was among the first to identify GABABR2 and describe the genomic organization of the human GABABR1 and GABABR2 genes.
Presynaptic GABAB receptors regulate the efficiency of neurotransmitter release while postsynaptic GABAB receptors regulate the ability of neurotransmitter-gated ion channels to produce responses that alter neuronal excitability. Expression of two subunits GABABR1 and GABABR2 is believed required for receptor function; yet, we have shown that the majority of striatal neurons express high levels of GABABR1 in the absence of GABABR2. Our laboratory has also shown that the GABABR1 isoforms (GABABR1a and GABABR1b) are expressed under control of alternative promoters in the GABABR1 gene. Our ongoing studies are focused on the genomic organization of the human GABAB receptor subunit genes, transcriptional regulation of the alternative GABABR1a and GABABR1b promoters, and GABAB receptor subunit targeting in hippocampal and striatal neurons that differ in their expression of endogenous GABAB receptor subunits. These studies will provide new insights into the mechanism by which alternative GABAB receptor subunit synthesis may influence the plasticity of neuronal networks. The ability to control the specific gene expression of the alternative GABAB receptor subunits may also provide a novel treatment option for epilepsy and drug addiction.