David H. Farb, Ph.D. - US grants

The fundamental goal of the proposed research is to elucidate mechanisms of receptor modulation, focussing primarily on the GABA/A receptor of the vertebrate central nervous system. The proposed study has two major parts. First, we propose to extend our preliminary observations that indicate that Ca2+ ions play a fundamental role in modulating the function of the GABA receptor, with the aim of determining the mechanism whereby Ca2+ influences GABA receptor function. In particular, we will confirm or reject a model in which calcium plays a role as an obligatory cofactor in the process of GABA stimulated C1- transport. Secondly, we will carry out electrophysiological and radioligand binding studies to test a theoretical model relating electrophysiological and biochemical parameters of modulator action. In particular, we will determine whether it is possible to predict efficacy of modulators of the GABA response on the basis of binding kinetics. Our long-term goals are to construct molecular models of the interaction of neuromodulators with the GABA/A receptor and to gain an understanding of the molecular underpinnings of GABA/A receptor function. These studies are expected to provide the basis for modeling of the relationship between the GABA receptor and its various modulatory sites, and may yield novel insights into the molecular mechanisms underlying the function of ligand-gated anion channels.

The ability of steroid hormones to influence profoundly the excitability of the CNS is well documented. The broad objective of this proposal is to explore pharmacologically the molecular mechanisms whereby steroids modulate CNS excitability. Abnormal activation of amino acid receptors has been proposed to play a role in the etiology of psychiatric disorders such as anxiety, depression and schizophrenia. Understanding the mechanisms of steroid actions on the CNS may lead to new strategies for the treatment of psychiatric disorders. During the course of our studies we came upon the unexpected finding that pregnenolone sulfate (PS), an abundant neurosteroid, acts as a positive allosteric modulator at the NMDA receptor while inhibiting the kainate, AMPA, glycine, and GABA responses. A major focus of the research plan will be to test our working hypothesis that steroids such as PS regulate the balance between excitation and inhibition on neurons derived from the vertebrate CNS by acting on excitatory and inhibitory amino acid receptors. Toward this end, whole-cell voltage-clamp and current-clamp techniques will be utilized to character electrophysiologically the effects of PS and related steroids on excitatory and inhibitory amino acid receptor-mediated responses of embryonic chick spinal cord neurons maintained in primary monolayer cell culture. The proposed study has three major parts: First, we will screen a series of steroids for activity on amino acid receptor-mediated currents. Secondly, we will determine the potency, efficacy, and mode of action for PS and each steroid identified, with the goal of elucidating the mechanism(s) of steroid action(s) through a study of their structure-activity relationships. In particular, we will focus our studies on PS and on other steroids that are active at NMDA and non-NMDA glutamate receptors. Finally, the effects of steroids on excitatory and inhibitory synaptic transmission at single synapses will be investigated. In the long run, we will determine whether chronic treatment with active steroids such as PS, progesterone, and reduced metabolites of progesterone induces functional changes of excitatory or inhibitory amino acid receptors and of synaptic transmission at identified synapses. These studies will provide a strong basis for evaluating the role of steroids as modulators of neuronal function, and a foundation for development of novel steroid-based drugs for the treatment of psychiatric disorders.

GABA receptor; behavioral genetics; protein deficiency; mother /embryo /fetus nutrition; mental retardation; hippocampus; genetic regulatory element; developmental neurobiology; neocortex; DNA binding protein; transcription factor; disease /disorder model; nucleic acid sequence; dietary proteins; behavioral /social science research tag; site directed mutagenesis; newborn animals; laboratory rat; transfection; human genetic material tag; embryo /fetus tissue /cell culture; RNase protection assay;

health care; technology /technique development; mental disorders; proteins; lipids; biomedical resource; Chordata; biological products; drug screening /evaluation; nervous system; psychology; behavioral /social science research tag;

DESCRIPTION (provided by applicant): The predoctoral Program in Biomolecular Pharmacology at Boston University School of Medicine was initiated in 1990 and received this NIGMS Institutional Training Grant in 1997. In the ensuing ten years of NIGMS support, this university-wide program has flourished, providing a unique learning environment for doctoral students that combines an innovative curriculum, interdisciplinary laboratory rotations, and expanded opportunities for thesis research by bridging multiple departments across Boston University's two campuses. Students enter the program from one of three academic units: Pharmacology and Experimental Therapeutics (PET), Biomedical Engineering (BME), and Molecular Medicine (MM). Program trainees in BME and MM experience an integrated curriculum designed to provide enriched training in pharmacology that is coordinated with specialized training in their discipline while trainees in PET gain access to diverse research and educational experiences that build upon those provided by core pharmacology faculty. The curriculum stresses fundamental pharmacologic principles as well as key issues governing interactions of bioactive molecules, challenges of drug delivery for novel therapeutics, animal models and their relevance to the clinic, and the challenges for modern drug discovery. Participating faculty, originally fifteen and now forty-six, contribute expertise in focus areas including neuropharmacology, vascular and cancer pharmacology, genomics, proteomics, animal models (transgenic and behavioral), structural biology, and DMA, RNA, and protein chemistry. Sites for thesis research are also located in departments of Chemistry, Biology, Psychology, Neurology and Psychiatry. A summer internship with collaborating scientists at Wyeth Research has been fully implemented and career guidance and mentorship/leadership opportunities engage trainees with quality experiences that are relevant to their future careers in pharmacology both in academia and industry. Since inception, forty-two students have been supported by this NIGMS program that was one of the first to provide an interface for quantitatively trained BME students to link their core studies with training in pharmacological sciences. The program has continued this mission and expanded to position students with intellectual tools needed to advance drug discovery, medicine, and the future of global health by training the next generation of leaders equipped to translate basic research advances into medications.

Ethanol exerts profound acute and chronic effects onreceptors for amino acid neurotransmitters in the CNS. In particular, acute exposure to ethanol potentiates the ganna-aminobutyric acid type-A receptor (GABAAR) mediated response, while chronic exposure alters the pharmacology and density of GABAARs in animal models and inpost-mortem brain from alcoholics. It has been proposed that chronic ethanol may cause a switch in the expression of receptor subunit mRNAs, producing an isoform(s) with altered pharmacolgical and physiological properties. However, the mechanism by which receptor function is altered remains unknown yet crucial to our understanding of ethanol tolerance and dependence. During the course of our investigation into the identification and function of promoters for human GABAAR subunit genes, we discovered that ethanol can stimulate (by about 20-fold) alpha1 promoter activity in transfected primary rat neocortical cultures, and that a 63 bp region located in the proximal promoter is crucial for ethanol regulation. The activity of the beta1 promoter is reduced by ethanol, suggesting that one or more cis-regulatory elements are involved in the cell's adaptive response to ethanol. A major objective of this project will be to test our working hypothesis that ethanol interacts with cell surface receptors and exerts genomic regulatory effects through a cellular signal transduction system(s), activating or inhibiting transcription factors which bind to ethanol reposive cis-elements. We have discovered that GABAAR gene diversity arose fromm the duplication and subsequent translocation of an ancestral human alpha-alpha-beta-gamma gene cluster, spawning the clusters on chromosomes 4, 5 and 15. It is our hypothesis that the close evolutionary relationsip of genes within and across clusters predicts coordinate regulation regulation of related genes in different clusters by ethanol. To examine the above hypotheses, ethanol responsive elements in alpha, beta and gamm subunit gene promoters will be identified functionally, using primary neuronal cultures transfected with luciferase reporter constructs containing various deletions and point mutations. We will compare the sequence and fucntion of ehtanol responsive elements from different GABAAR subunit promoters to identify conserved regulatory regions and shared transcription factors. Inhibitors and activators of intracellular messengers will be used to determine the cellular response system that couples receptor modulation to transcriptional regulation. Collectively, these experiments will add to our understanding of the neuron's adaptive response(s) to chronic ethanol exposure, and, in the long-run to the development of diagnostiv and therapeutic agents.

DESCRIPTION (provided by applicant): The mesotelencephalic dopamine system is implicated in the etiologies of schizophrenia and Parkinson's disease as well as in the reinforcing properties of psychostimulants, including cocaine and amphetamine. Recent evidence from our laboratory indicates that a number of neuroactive steroids influence dopamine transmission in the striatum by potentiating or inhibiting ionotropic glutamate receptor function. The major objective of this project is to follow up on these findings by assessing the activity of these neuroactive steroids in the nucleus accumbens, the limbic portion of the striatal complex that is the locus for the reinforcing effects of many drugs of abuse. To that end, we will assess the modulatory influence of neuroactive steroids on the behavioral and neurochemical effects of ionotropic glutamate receptor agonists in the core and shell of the nucleus accumbens. Using microdialysis (coupled with HPLC-EC and HPLC-MS) and behavioral techniques we will determine, i) the accumulation of systemically administered neuroactive steroids in the brain, ii) the behavioral and neurochemical consequences of neuroactive steroid modulation of ionotropic glutamate receptors in the nucleus accumbens shell, iii) the influence of neuroactive steroids on the initiation of behavioral sensitization to cocaine and iv) the influence of neuroactive steroids on cocaine priming-induced reinstatement of cocaine-seeking behavior. Collectively, these experiments will provide information on the activity of neuroactive steroids in the CNS, which will be invaluable for the development of steroids as potential pharmacological treatments for disorders involving ionotropic glutamate receptors in general and the craving associated with cocaine addiction in particular.

[unreadable] DESCRIPTION (provided by applicant): N-methyl-D-aspartate receptors (NMDARs) play a crucial role in cognition and long-term memory, and have been implicated in a variety of pathological conditions, including stroke, intractable pain, neurodegenerative diseases, and spinal cord injury. We have shown that endogenous neurosteroids, such as pregnenolune sulfate (PS), can specifically modulate NMDA receptor function. Evidence for at least two neurosteroid recognition sites has emerged, one associated with positive modulation and the other with negative modulation. We have demonstrated recently that the direction of modulation by PS is dependent upon which of the NMDA receptor Type-2 subunits is present. NR2A or NR2B confers PS potentiation, whereas NR2C or NR2D confers PS inhibition. To define the structural determinants of receptor subtype-specific neurosteroid modulation, multiple chimeras from NR2B and NR2D parent cDNAs were constructed and analyzed by voltage clamp electrophysiology in Xenopus oocytes containing transiently expressed receptors. We have identified a Steroid Modulatory Domain 1 (SMD1) on NR2B, containing the fourth transmembrane segment (M4) and part of the adjacent M3-M4 linker region. SMD1 is required for rapid potentiation of the NMDA response by PS. In addition to the rapid direct modulation of NMDAK by neurosteroids, there is a slow phase of modulation that is mediated by PKC. The proposed studies build on and extend these findings. [unreadable] [unreadable] Using site-directedmutagenesis of NR2B/2D and NR2D/2B chimeric subunits to eliminate and then reinstate PS modulation, specific residues that contribute to the response at different NMDAR subtypes will be identified. Cysteine substitution and sulflaydryl-modifying reagents will be used to determine whether these residues are solvent accessible and participate inPS action. Results of these studies will be used to formulate a pharmacological model of NMDAR modulation by neurosteroids. In addition, the possible interactions between the steroid, zinc, and proton modulatory sites will beinvestigated. We will also investigate the mechanism of slow potentiation by PS, which, in contrast to rapid potentiation, requires the action of cellular signal-transduction systems. We will determine whether the domain(s) that control slow potentiation are the same or different from those that control fast potentiation, and will investigate the contributions of rapid and slow potentiation to enhancement of excitotoxicity by PS in neurons and transfected HEK cells. These studies will enhance our understanding of the structural basis that underlies the specificity of neurosteroid modulation at NMDAP, s, and will provide the tools with which to study the biological and pharmacological relevance of endogenous neurosteroids in the nervous system. [unreadable] [unreadable]

Abstract: The devastating progression of Alzheimer's disease (AD) from the early prodromal to late stages has stimulated a search for drugs that prevent this progression and a quest for drugs that can treat the symptoms of memory loss to improve the quality of life even in the face of decline. We hypothesize that determining the effect of AD progression on the dynamic activity of important local circuits in the rodent hippocampus, the tri-synaptic circuit known to underlie spatial memory, will produce an early indicator of brain dysfunction that is more relevant for the discovery of drugs that will work in humans, especially given the conservation of this circuit in all mammals. The firing patterns of CA3 & CA1 pyramidal cells (or ?place cells?) within the hippocampus respond to and encode spatial representations. Our previous in vivo electrophysiological study of aged impaired rats, a model for amnestic cognitive impairment (aMCI), demonstrated that we are able to quantify age and novelty specific effects of drugs on CA3 & CA1 place cell dynamics and on spatial memory (Robitsek 2015). In this application, we propose to use an exciting new rat model for AD (TgF344-AD) that displays amyloid plaques and, importantly, neurofibrillary tangles to determine how the progression of pathology seen in human patients is associated with alterations in hippocampal place cell dynamics over time. In addition, we will probe the acute effects of a GABA- A receptor negative allosteric modulator selective for tonically active alpha5 subunit containing receptors that is a memory enhancer on place cell dynamics. We will use high density in vivo electrophysiology for monitoring ?place cell? function with age in this novel rat transgenic model to ask the following questions: When does the tri- synaptic circuit become disturbed? Is early disturbance associated with P-tau and neurofibrillary tangle formation, or is it independent? Is there a distinct part of the hippocampal tri-synaptic circuitry that is most vulnerable during disease progression? And finally, can we begin to explain the mechanism by which a negative allosteric modulator of active inhibitory receptors results in spatial memory enhancement. We anticipate that completion of these studies will identify the activity profile of CA3 & CA1 place cells in AD rats that eventually leads to a loss in memory and spread of neuropathology. These studies will be a first step toward elucidating the onset of neural circuitry dysfunction underlying spatial memory decline over a human APP695 and PS1 background that exhibits progressive neurofibrillary tangles and cognitive impairment and will provide a foundation for improved assessment for acute administration of memory enhancers in AD management.

Major barriers impede the translation of basic research findings from preclinical animal models of Alzheimer's disease (AD) into the discovery of methods for early detection of AD onset and the treatment of memory dysfunction. We hypothesize that early dysfunction of memory will be observable as dysfunction of hippocampal trisynaptic circuit dynamics (TCDs) that are specifically associated with spatial memory. The individual firing patterns of CA3 & CA1 pyramidal cells (or ?place cells?) within the trisynaptic circuit can be measured while acquisition, encoding, recall, and reconsolidation of spatial representations occurs. Our recent in vivo studies of aged animals, a model for amnestic mild cognitive impairment, have revealed age and novelty specific effects on CA3 & CA1 place cell dynamics that are rapidly reversed by acute administration of levetiracetam + valproic acid (Robitsek 2015). We also hypothesize that changes in properties of single neurons within the trisynaptic circuit will be an early signature for future behavioral impairment and for identifying genome responses that may be most relevant to AD during the prodromal phase. In this multidisciplinary application, we propose to use the novel TgF344-AD rat model (expressing mutant human amyloid precursor protein (APPsw) and presenilin 1 (PS1?E9)) to identify hippocampal TCDs that change during development of AD-associated memory dysfunction and to interrogate their underlying transcriptome and methylome. Prodromal changes in neural activity appear to play a role in the progression of neuropathological changes observed in AD by promoting the release of tau and the formation of neurofibrillary tangles. Accordingly, plasma tau levels are associated with cognitive decline and conversion of mild cognitive impairment into dementia. Less is known, however, about the cell specific response of the genome during the onset and course of AD progression that may factor significantly into disease etiology and resilience, nor the composition of the transcriptome of individual cells that respond dynamically to the onset and growing burden of inflammation. As a first step to fill this gap in knowledge, we propose the following two Aims: 1) To determine the temporal window for altered neural network activity in TgF344-AD rats that anticipates the age related progressive deterioration of spatial memory (in vivo electrophysiology); and 2) To determine the transcriptome of unique cell types in the TgF344-AD hippocampus (single cell RNA-sequencing (scRNA-seq) using a within-subjects design (Aim 1)) and the state of the TgF344-AD hippocampal genome as determined in parallel molecular studies (bulk RNA-seq and Methyl-seq). The proposed research has the potential to detect prodromal signatures of genomic events that underlie the onset of memory decline, uncovering relevant molecular determinants that may enable interventions to enhance memory and, conceivably, slow disease progression and human suffering.