Gerard Sanacora - US grants

depression; electroconvulsive therapy; gamma aminobutyrate; cerebral cortex; neurobiology; clinical research; human subject;

DESCRIPTION: (adapted from applicant's abstract): I have been fortunate to work in stimulating environments that have afforded me substantial opportunities to engage in research training throughout my graduate (M.D., Ph.D.) and residency training. In July, I will continue my training an additional year with the Daniel X. Freedman Clinical Neuroscience Fellowship at Yale University. However, without support from a K08 award, I would be required to assume substantial clinical responsibilities in order to support my salary when I join the Yale faculty in July, 1999. I have designed this K08 award to enable me to pursue a comprehensive five-year research-training Plan. This plan will provide me with the background and skills necessary to become an independent investigator-using novel magnetic resonance spectroscopy (MRS) techniques. I would like to apply these techniques to the study of GABA and glutamate neurotransmission related to the pathophysiology and treatment of affective disorders. Over the past two years I have developed basic skills in-proton-MRS (1H-MRS). We have produced exciting preliminary data describing reductions in cortical GABA associated with depression, and elevation of cortical GABA levels following electroconvulsive therapy (ECT). I propose to continue these investigations with two aims. 1) To fully characterize both GABAergic and glutamatergic systems in affective disorder disease states. (Years 1-3) and 2) To use pharmacological probes along with [13C]-MRS to examine possible pathophysiological mechanisms related to amino acid neurotransmitter regulation in depression. In order to advance the use of MRS applications in the study of affective disorders, I have identified several key areas where I will require additional training including: 1) nuclear magnetic resonance, 2)1 neurochemistry, kinetic modeling, and related neuroimaging technologies, 3) clinical and basic neuropharmacology applicable to affective disorders research; and 4) clinical trials methodologies. I believe my background in the basic sciences will enable me to complete the rigorous training plan outlined in this application. This training along with continued practical application of the technology under the co-mentorship of Drs. Rothman and Krystal should thoroughly prepare me for a career as an independent investigator using MRS technology to study brain neurochemistry. This novel line investigation will open previously inaccessible areas of research, with a broad range of future applications.

DESCRIPTION (provided by applicant): Several lines of evidence suggest amino acid neurotransmitter system abnormalities contribute to the neurobiology of depression. In humans, there is clear evidence of altered amino acid neurotransmitter function in depressed individuals. Most notably, depressed patients appear to have widespread GABA reductions, with studies consistently demonstrating lower plasma and CSF GABA concentrations in depressed subjects compared to non-depressed controls. More recently, magnetic resonance spectroscopy also demonstrated significantly decreased GABA concentrations in the occipital cortex of depressed patients. These reduced GABA concentrations appear to be present in a large subgroup of depressed subjects suggesting that it may be a useful marker in identifying a subtype of depressed individuals sharing a common pathophysiology. Furthermore, reduced GABA concentrations appear to normalize following ECT or antidepressant medications, suggesting a possible association to the antidepressant effects of the treatment modalities. Other evidence suggests that altered glutamatergic function is also associated with the pathogenesis and pathophysiology of mood disorders. Consistent with this hypothesis, elevated cortical glutamate concentrations were recently demonstrated in the same group of depressed subjects as those showing reduced GABA concentrations. The principal objective of this study is to gain additional information regarding the underlying neurobiological mechanism responsible for the abnormalities in amino acid neurotransmitter concentrations in the occipital cortex of depressed subjects. We plan to achieve this objective by using 1H-MRS and 13C-MRS to obtain measures of the amino acid concentrations, rates of GABA synthesis and glutamate/glutamine cycling in medication-free depressed patients and healthy comparison subjects. In addition, we will continue to investigate the potential association between cortical GABA concentrations and both the various clinical subtypes of depression and allelic variants of the GAD65 gene.

DESCRIPTION (provided by applicant): This KO2 application seeks support that is essential for Dr. Sanacora's continued career development as a newly-independent clinical researcher. Funded by a NIMH K08 mentored career award (K08-MH-01715) Dr. Sanacora has attained considerable expertise in the use of magnetic resonance spectroscopy as a tool to investigate the role of amino acid neurotransmitter systems in the pathophysiology and treatment of mood disorders over the past 5 years. Thanks to the mentorship Dr. Sanacora received and the excellent environment provided to him through the Magnetic Resonance Center and the Department of Psychiatry at Yale he demonstrated considerable research productivity during this time as evidenced by his peer-reviewed publications and a recently-awarded RO1 (MH071676-01, G. Sanacora, P.I.). However, this K08 expired 6/31/04. This KO2 award is vital for sustaining Dr. Sanacora's current full-time research focus and for assuring his successful matriculation into a fully independent investigator. Justification for the award is provided by a comprehensive 5-year Career Development Plan that builds on his current expertise in the study of amino acid neurotransmitter contributions to mood disorders and the MRS methodologies used to investigate this area. Specifically, his goals are to acquire specialized skills in several additional areas that will make it possible for him to extend this work into areas of more direct scientific and clinical significance: 1) 13C-glucose MRS methods to measure rates of synthesis and cycling, 2) 13C-acetate studies to probe glial cell involvement in mood disorder pathology, 3) clinical trial experience to allow him to address the potential clinical usefulness of drugs targeting the amino acid neurotransmitter systems, and 4) basic molecular neurobiology related to amino acid neurotransmission that will enable him to continue to conduct relevant translational research in mood disorders. Intensive training is provided by an integrated curriculum of intramural coursework, extramural didactics, individualized preceptorships, interactive symposia, and research-related organizational meetings. Three representative studies are included to illustrate how the development of these skills will be used to advance the line of research that was initiated in his previous K08 award into the realms of pathophysiology and clinical practice. Thus, receiving this award would allow him the opportunity to improve the basic understanding of how the amino acid neurotransmitter systems and glial cell pathology contribute to the neurobiology of mood disorders. This work has direct clinical relevance since it is likely to lead to the development of novel diagnostic methods and treatment modalities for these disorders.

6. PROJECT SUMMARY/ABSTRACT Increasing evidence suggests a tight relationship exists between stress, amino acid neurotransmitter systems and Major Depressive Disorder (MDD). The relationship between stress and glutamatergic signaling is illustrated by the finding of elevated extracellular glutamate (Glu) concentrations in several brain regions following stress exposure. A recent series of studies demonstrate that several classes of Glu-modulating agents possess antidepressant-like properties in animal models, as well as in patients with mood disorders, thus providing additional support for the connection between Glu neurotransmission and MDD. Based on findings of abnormal GABA and Glu concentrations in individuals diagnosed with MDD, and a growing literature documenting significant reductions in the glial cell number and density associated with MDD, we have proposed a model whereby impaired astrocyte function and a disruption of glutamate/glutamine cycling serves a central role in the pathophysiology of MDD. Riluzole, an FDA-approved Glu-modulating agent used to delay ALS progression, exhibits anti-glutamatergic properties via modulation of neuronal Glu release and enhancement of Glu uptake. Several studies now also suggest a therapeutic action of riluzole in patients with MDD. Consistent with our hypothesized model, recently collected preliminary data from our laboratory demonstrate that riluzole attenuates and reverses the effects of behavioral stress in several rodent models that are commonly used to test for antidepressant drug activity. In addition, other work from our group using 13C- MRS to measure the rate of Glu, glutamine (Gln), and GABA synthesis, suggests that chronic stress reduces astrocyte metabolism and Glu/Gln and GABA/Gln cycling. Riluzole admnistration resulted in increased rates of Glu/Gln cycling and attenuates the effects of stress on astrocyte function. To further test the hypothesis that enhanced Glu uptake could prevent or attenuate the effects of stress and have antidepressant-like activity, we recently completed a study examining the effects of ceftriaxone, a ¿-lactam that results in increased expression of the Glu transporter GLT1 and increased Glu uptake, in several rodent models used to assess antidepressant-like activity. The studies demonstrated that ceftriaxone has a profile consistent with antidepressant agents. We would now like to examine the effects of chronic stress and Glu-modulating drugs on Glu cycling, extracellular Glu content, and the relationship between these effects on glutamatergic neurotransmission and animal models of depression. We will also specifically explore the role of GLT1 mediated Glu uptake in stress and the mechanism of these drugs using pharmacological inhibitors of glutamate uptake and a genetic model of impaired GLT1 function in mice. The results of these studies could significantly expand our understanding relating the physiological response to stress to mental illness. Moreover, the results of the studies could potentially provide us with novel targets for antidepressant drug development.

DESCRIPTION (provided by applicant): This collaborative R01 investigates the safety and efficacy of the glutamate-modulating and neuroprotective agent riluzole in a randomized double-blind placebo-controlled clinical trial (RCT) in patients with treatment- resistant depression (TRD). The recent findings of the STAR*D study highlight the fact that our current armamentarium of antidepressant medications, developed out of the monoamine hypothesis, has serious limitations. There is now emerging evidence that amino acid neurotransmitter (AANt) systems may also contribute to the pathophysiology of major depressive disorder (MDD), and that drugs targeting these systems may have potent antidepressant properties. Riluzole is currently approved by the US FDA for the treatment of amyotrophic lateral sclerosis (ALS). It has been shown to have a variety of neurobiological effects on glutamatergic function associated with the drug's neuroprotective and plasticity-enhancing properties. There have now been two recent open-label studies showing riluzole to be effective in TRD as well as several reports demonstrating riluzole's efficacy in bipolar depression, generalized anxiety disorder, and obsessive-compulsive disorder. However, no RCT has been performed in TRD. This randomized, double-blind, placebo-controlled trial, using a sequential parallel comparison design, evaluates the efficacy and safety of riluzole augmentation of the selective serotonin reuptake inhibitor citalopram in outpatients ages 18-65 with moderate TRD. Significance to Public Health: In this unique 5-year proposal, investigators from four institutions [Yale, MSSM, MGH and NIMH (MAP)] will conduct a RCT to examine the efficacy, safety and tolerability of adjunctive riluzole treatment in TRD. Demonstration of riluzole's benefit would represent a major advance for patients with difficult-to-treat depression, and may help elucidate our understanding of the pathophysiology of MDD and the mechanism of antidepressant action. Most immediately, since riluzole is already a FDA-approved medication that has been safely prescribed to thousands of patients with ALS, positive findings from this study could rapidly disseminate into clinical practice and would encourage further investigation of this strategy. Demonstrating riluzole's efficacy in TRD would open the door to the discovery of future medications targeting the glutamatergic system that could be used to fight this common and devastating disorder.

DESCRIPTION (provided by applicant): Evidence of altered brain glutamatergic and GABAergic function is reported in a wide array of psychiatric and neurological disorders. Most current treatments for neuropsychiatric illness target the monoamine systems and have limited efficacy. The acknowledgement of this fact has led to an increased drive to develop novel drugs acting through alternative mechanisms. There is now intense focus on the amino acid neurotransmitter systems (glutamate/GABA/glutamine) as targets for treatment, creating the need to identify reliable biomarker assays. In vitro cell culture and brain slice preparations often fail to predict in vivo responses to glutamate- modulating drugs in humans or unanesthetized animals. There is a pressing need for quantitative assays of glutamate/GABA neurotransmission that reflect the in vivo physiological state, avoids anesthesia or postmortem effects, and can be translated more directly to humans. Recently, a novel ex vivo 3D in situ magnetic resonance spectroscopic imaging (MRSI) approach was introduced, which generates high spatial resolution quantitative maps of numerous neurochemicals from the brain's of rodents euthanized by microwave irradiation, preserving neurochemical levels and microstructure. Applied with 13C labeled tracers, high-spatial resolution 2D and 3D maps of 13C-labeled amino acids can be generated. Appropriately validated, rate maps reflecting neuronal (glutamatergic and GABAergic) and astroglial metabolism, and neurotransmitter cycling can be extracted from the data sets. Combined with other neuroimaging modalities (e.g., T1, T2 diffusion-tensor), quantitative measurements of high information content of multiple endpoints for metabolism, structure, and connectivity can be obtained. The addition of other '-omics' end-point measurements are also possible. With all neuroimaging efficiently acquired in the same brain and coordinate space, the proposed assay has significant potential to reveal altered glutamatergic/GABAergic neuronal and glial pathways, accelerating preclinical drug evaluation and treatment response. Development of this methodology would afford investigators opportunity to obtain thousands of precisely defined neurochemical and anatomical data points in a single experiment, in contrast to present methods (e.g., cell-free extracts or tissue slices) which access only one or a few regions at a time. Aim 1 will develop and validate the ex vivo metabolic flux mapping assay against 13C fractional enrichment measured in cell-free extracts, and evaluate the accuracy of the ex vivo flux measurements against in vivo MRS time courses. A double-labeling approach to increase reliability and efficiency will also be evaluated. Aim 2 will develop automated metabolite quantification and analysis methods for the ex vivo 3D MRSI/MRI high density data sets for efficient and unbiased extraction of available information, enhanced data quality, and greatly increased throughput. Aim 3 will apply the ex vivo flux mapping assay to characterize and compare the acute effects of glutamate modulating drugs with a broad range of potential therapeutic effects, on regional rates of glutamate/GABA neurotransmission with receptor activated signaling (phosphoproteins).