Chang-Gyu Hahn - US grants

DESCRIPTION: (Adapted from applicant's abstract) This project focuses upon a pathophysiologic study of bipolar disorder using olfactory receptor neurons (ORNs) from patients. ORNs, the only central nervous system (CNS) neurons that are obtainable through olfactory epithelial biopsy, can be a powerful tool for the molecular study of bipolar disorder. ORNs, accessible even multiple times in the same individuals, enables characterizing the neuronal pathology in a state- vs. trait dependent manner. Furthermore, since ORNs propagate in tissue culture condition, an in vitro cellular model of the illness can be developed. Recent molecular studies have implicated various abnormalities in signal transudation mechanisms and gene regulation in bipolar disorder. However, the pathophysiologic interpretation of these findings has been difficult, because it requires an examination of the proposed molecules in the neuronal tissues from patients in different phases of the illness. Recently, the investigators have investigated calcium homeostasis in ORNs from bipolar patients. The intracellular calcium responses in ORNs from 7 medication free euthymic bipolar patients were strikingly different than healthy control subjects. These suggest that ORNs from bipolar patients may express trait dependent intracellular abnormalities of the illness. In this study, characterization of the odorant induced intracellular calcium response in ORNs from depressed, hypomanic/or manic and euthymic bipolar patients are proposed. If the intracellular characteristics in vivo are maintained in vitro, cultured ORNs can be an in vitro model of the illness. To examine if the intracellular characteristics in vivo are maintained through time in tissue culture, odorant induced intracellular calcium responses will be characterized in cultured ORNs from euthymic patients, in comparison to acutely dissociated ORNs from the original tissue. ORNs provide a unique opportunity to characterize neuronal gene regulation in a state dependent vs. trait dependent manner. By applying single cell molecular techniques in conjunction with calcium imaging of odorant responsiveness, the investigators will be able to investigate gene regulation in functionally defined neurons from patients. In this proposal, the feasibility of amplifying anti-sense RNA from the ORNs after odorant responsiveness has been characterized by calcium imaging will be established.

DESCRIPTION: (Adapted from applicant's abstract) This project focuses upon a pathophysiologic study of bipolar disorder using olfactory receptor neurons (ORNs) from patients. ORNs, the only central nervous system (CNS) neurons that are obtainable through olfactory epithelial biopsy, can be a powerful tool for the molecular study of bipolar disorder. ORNs, accessible even multiple times in the same individuals, enables characterizing the neuronal pathology in a state- vs. trait dependent manner. Furthermore, since ORNs propagate in tissue culture condition, an in vitro cellular model of the illness can be developed. Recent molecular studies have implicated various abnormalities in signal transudation mechanisms and gene regulation in bipolar disorder. However, the pathophysiologic interpretation of these findings has been difficult, because it requires an examination of the proposed molecules in the neuronal tissues from patients in different phases of the illness. Recently, the investigators have investigated calcium homeostasis in ORNs from bipolar patients. The intracellular calcium responses in ORNs from 7 medication free euthymic bipolar patients were strikingly different than healthy control subjects. These suggest that ORNs from bipolar patients may express trait dependent intracellular abnormalities of the illness. In this study, characterization of the odorant induced intracellular calcium response in ORNs from depressed, hypomanic/or manic and euthymic bipolar patients are proposed. If the intracellular characteristics in vivo are maintained in vitro, cultured ORNs can be an in vitro model of the illness. To examine if the intracellular characteristics in vivo are maintained through time in tissue culture, odorant induced intracellular calcium responses will be characterized in cultured ORNs from euthymic patients, in comparison to acutely dissociated ORNs from the original tissue. ORNs provide a unique opportunity to characterize neuronal gene regulation in a state dependent vs. trait dependent manner. By applying single cell molecular techniques in conjunction with calcium imaging of odorant responsiveness, the investigators will be able to investigate gene regulation in functionally defined neurons from patients. In this proposal, the feasibility of amplifying anti-sense RNA from the ORNs after odorant responsiveness has been characterized by calcium imaging will be established.

DESCRIPTION: (provided by applicant) This proposal seeks a structured research training program, which will enable Chang-Gyu Hahn. M.D. Ph.D. to become an independent investigator as he demonstrates the utility of olfactory receptor neurons (ORNs) to examine the neurobiology underlying bipolar disorder. Career Goals and Objectives: With the career goat of being able to apply the most advanced molecular techniques to clinically relevant research paradigms through the ORN system, he proposes a career development plan focusing on following areas. 1) ionic imaging and the neurobiology of the human ORN system and 2) Single cell antisense RNA amplification technique. Career Development Activities: The candidate will receive specialized training in ionic imaging in human olfactory neurons (guided by Nancy Rawson, PILD, co-mentor) and in single cell molecular technique (supervised by James Eberwine, Ph.D., co-mentor). Dr. Karl Rickels will provide individual supervision for the treatment outcome study component of the specific aim 2. Dr. Robert H. Lenox will oversee the career development program through integrating formal courses, lab meetings and supervisions. Background: ORNs, the only CNS neurons that are obtainable through olfactory epithelial biopsy, provide a unique opportunity to investigate molecular events in neurons from living subjects. By obtaining ORNs during specific stages of the illness, in conjunction with a longitudinal follow-up of patients, pathophysiologic significance of molecular events in neurons can be linked to bipolar disorder. Hypothesis: Our preliminary data suggest that intracellular calcium responses to odbrants are altered in bipolar disorder. I) Intracellular calcium responses of ORNs to odorants are predominantly a decrease as a trait of bipolar disorder. 2) The decreased ICa responses in ORNs from bipolar patients are due to altered expression of the genes that are involved in calcium flux. 3) The percentage of ORNs that respond to odorant stimulation is overall decreased following lithium treatment. 4) ORNs in explant cultures from bipolar patients will show similar ICa responses as in vivo ORNs of patients. Specific Aims: 1) To characterize trait- and mood state- dependent alterations in ICa homeostasis and gene regulation in ORNs from bipolar patients. 2) To identify therapeutically relevant changes in ICa homeostasis following lithium treatment. 3) To characterize ORN cultures from bipolar patients using odorant induced ICa responses.

[unreadable] DESCRIPTION (provided by applicant): Recent molecular genetics studies implicate neuregulin 1 (NRG1) and its receptor, erbB, in the pathophysiology of schizophrenia. Among NRG 1 receptors, erbB4 is of particular interest because of its crucial roles in neurodevelopment and in modulation of N-methyl-D-aspartate (NMDA) receptor signaling. We have recently examined the expression and transduction of the NRG1- erbB4 mechanism in the postmortem brains of schizophrenia and control subjects. Using a novel postmortem tissue stimulation approach, we found striking alterations of NRG1- erbB4 signaling in the prefrental cortex of schizophrenia subjects. First, NRG1-induced erbB4 activation, measured by tyrosine phosphorylation of erbB4 and its association with PSD-95, was dramatically enhanced, while the expression levels of NRG1 or erbB4 were not altered. Second, the association of erbB4 with PSD-95 and NMDAR as well as PSD-95's coupling with NMDAR were also significantly altered in schizophrenia. Third, NRG1 stimulation attenuates NMDAR activation in the human prefrontal cortex as shown in rodents. NMDAR activation, measured by tyrosine phosphorylation of the receptors, was significantly attenuated in schizophrenia subjects, which we believe to be the first direct demonstration of NMDAR hypofunction in the brains of patients. Finally, when the brain tissues were co-stimulated with NMDA and NRG1, NRG1 induced NMDA attenuation was even greater in schizophrenia subjects, suggesting that the dyregulated NRG1 -erbB4 signaling in schizophrenia may contribute to NMDAR hypofunction. Our central hypothesis is that altered erbB4 signaling in schizophrenia is associated with altered protein -protein interactions in the PSD, including NMDAR complexes. The aims of this proposal are designed to test a model in which altered erbB4 - postsynaptic density (PSD) protein association in schizophrenia leads to enhanced erbB4 signaling, which in turn results in NMDAR hypofunction. By doing so, we will be able to explore whether dysregulated protein - protein interactions in the PSD is a pathophysiologic mechanism for schizophrenia. Aim 1 will first assess whether hyperactive erbB4 signaling is brain region- or ligand-specific in schizophrenia. Aim 2 will specifically address protein protein interactions among erbB4 and other PSD proteins. Aim 3, will further characterize the impact of erbB4 dysregulation on NMDAR signaling and explore possible mechanisms. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): As many as a third of persons with bipolar disorder (BD) are clinically unresponsive to treatment with mood stabilizers and thus suffer a devastating clinical course. Studies over decades, in search of molecular targets of mood stabilizers, have demonstrated that mood stabilizers, lithium in particular, robustly regulate various intracellular signaling mechanisms. It is still unknown, however, whether these drugs regulate intracellular signaling mechanisms in human neurons or if such regulation actually contributes to therapeutic efficacy. We propose to address these issues by testing the effects of lithium on neural tissues obtained from biopsies of the olfactory neuroepithelium (OE) of bipolar disorder (BD) patients. Several dimensions of biological processes may mediate intracellular signaling and complex behavioral manifestations such as therapeutic efficacy: signaling -> gene regulation -> cellular outputs -> neuronal functions -> mood states. To test therapeutic relevance of these biological processes, we will assess BD patients' OE biopsy tissues and relate intracellular signaling mechanisms, gene regulation and neuronal function to their clinical responsiveness to lithium treatment. To that end, we will recruit BD patients in a pair of clinical studies; one cross-sectional and one prospective. The cross sectional study (Aim 1a, Group I patients) will test molecular and cellular parameters in OE tissues as a molecular profile that influences the patient's response to lithium across their lifetime. The prospective study (Aim 1b, Group II patients) will test the relationships between signaling modulation and improvement of acute mood episodes. To accomplish the objectives of Aim 1, we will examine OE tissues of patients immediately after biopsy (ex vivo paradigm) to capture neuronal signaling at the time of procedure. To trace these signaling changes to their cellular outputs or neuronal modulation, we will examine gene transcription and upstream molecular events. Using an in vitro OE study paradigm, we will conduct a detailed analysis of upstream molecular events and downstream transcriptional regulation (Aim 2). To further connect these molecular events to cellular functions in the context of therapeutic efficacy, we will examine neurotrophic/protective effects in the OE tissues of Group I and Group II patients. In addition, we will test BDNF or NMDA receptor transmission as molecular mechanisms underlying neurotrophic/protective effects using in OE cultures (Aim 3). This project will, for the first time, attempt to connect the dots in the cascade of signaling -> neuronal functions -> treatment response using patients' neuronal cells and, as such, will address molecular and cellular pathways and specific mechanistic hypotheses regarding the neurobiological basis of treatment resistance in BD.

DESCRIPTION (provided by applicant): Among the sensory modalities, olfaction is most closely associated with the temporo-limbic and frontal regions most commonly implicated in schizophrenia pathology. Olfactory probes may therefore be ideal tools to assess the structural and functional integrity of these neural substrates. To the extent that peripheral sensory afferents are disrupted in schizophrenia, the olfactory system - owing to its strategic anatomic location - may also be especially vulnerable to disruption. So, olfactory dysfunction may be a sensitive indicator of schizophrenia pathology, and may even serve as an early warning sign of disease vulnerability or onset. In addition, since neurogenesis and neurodevelopment persist throughout life in the olfactory neuro-epithelium and the olfactory bulb (OB), probes of the olfactory system may provide insight into the neurobiological basis of aberrant neurodevelopment. This project, now in its eleventh year, represents one of the only systematic efforts to characterize schizophrenia olfactory deficits from a neurobiological, rather than a simply behavioral, perspective. Recent studies have identified specific abnormalities implicating abnormal G protein-mediated signal transduction in peripheral olfactory receptor neurons (ORNs) and altered synaptic connectivity between ORNs and olfactory bulb as specific neurodevelopmental mechanisms that may be disrupted. This renewal application proposes a unique in vivo - in vitro translational investigation of these abnormalities. Detailed behavioral and electrophysiological studies of the peripheral olfactory system, including both epithelial and primary sensory cortical responses, will be conducted in 35 adult schizophrenia patients, 35 adolescents with a genetic risk for schizophrenia, 35 clinical-risk adolescents with prodromal symptoms, and appropriate age- matched control groups. Olfactory epithelial biopsy tissue will be acquired from the schizophrenia patients and age-matched controls. As the only accessible source of regenerating neural cells, olfactory epithelial biopsy offers an unparalleled opportunity to directly assess neuronal integrity in a living human. The epithelial biopsy tissue, along with olfactory bulbs obtained from post-mortem material, will used to investigate the cellular and molecular mechanisms that underlie the dysregulations observed in vivo. A particular focus will be G protein- mediated intracellular signal transduction and ORN-OB synaptic connectivity. The overarching hypothesis is that olfactory deficits reflect genetically-mediated abnormalities in intracellular signaling and neuron-to-neuron connectivity, which arise from developmentally disturbed processes of neurogenesis and synapse formation.

Accumulating evidence suggests that negative symptoms of schizophrenia may be mediated by altered circuit activity in the amygdala; specifically the basolateral amygdala (BLA) -nucleus accumbens (NA) and BLA- central nucleus (CeA) connections. Given that circuit activity is governed by synaptic strength/plasticity, NMDA receptor function, a key modulator of synaptic plasticity, may play a pivotal role in the BLA-NA and BLA-CeA circuitry. NMDA receptor function is linked to an array of neurocognitive functions and can induce endophenotypic behaviors of schizophrenia when perturbed in specific brain regions. We postulate that altered synaptic connectivity associated with dysregulated NMDA receptor function in the BLA-NA and BLA- CeA circuitry may lead to negative symptoms of schizophrenia. The BLA circuitry receives robust Dl and D2R mediated input from the prefrontal cortex and striatum and its overall activity is regulated by feedfoward and feedback inhibition via GABAergic neurons in intercalated islands. Cell type specific assessment of dopaminergic and NMDA receptor signaling therefore can reveal the intercellular dynamics leading to faulty circuitry of the amygdala in schizophrenia. To address this, we propose a postmortem study in which we conduct histologic and biochemical assessment of the synaptic strength and signaling activity of NMDA and dopaminergic receptors in a cell type specific manner. In the postmortem amygdala of 30 schizophrenia subjects and their matched controls, we will examine dendritic spine density and the integrity of synapses in principal and interneurons separately. Evaluation of receptor signaling in postmortem brains has been a challenging task. We have recently developed a number of paradigms that permit us to do so and found attenuation in tyrosine phosphorylation of NMDAR2A/2B reduced Src activity and altered protein interactions of NMDA receptor complexes in the PFC of SCZ patients. In this project we will examine the NMDA receptor function using immunoprecipitation -SRM-Mass spec analysis. Dopaminergic signaling can impact on NMDA receptor signaling in principal and interneurons modulating the overall circuit activity. We will examine presynaptic and postsynaptic segments of dopaminergic input using histologic and biochemical assessment.

DESCRIPTION (provided by applicant): The purpose of this application is to acquire a mass spectrometer, specifically the TSQ Vantage Triple Stage Quadrupole instrument from Thermo Scientific. The TSQ Vantage has unique capabilities to support ongoing studies that investigate pathophysiological pathways of diverse medical illnesses as well as programs that are developing novel therapeutics. This instrument will be used exclusively for quantitative proteomic applications by 10 NIH funded investigators located primarily at the Translational Research Laboratories (TRL) in the University of Pennsylvania. Ongoing projects initiated by these investigators have employed genomic discovery, biochemical, and molecular approaches to discover proteins of interest, posttranslational modifications, and protein networks that execute biological functions. The majority of these projects have advanced past the stages of discovery and specific and practical protein and peptide targets have been identified. A critical next step for these projects is to explore these discoveries in depth by quantifying changes in proteins and peptides that occur in various diseases or treatments. This goal cannot be accomplished with typical antibody based western blot, ELISA or even with currently available linear ion trap mass spectrometers due to lack of sensitivity, specificity, and/or high throughput of these approaches. Targeted peptide quantification is a highly selective and sensitive methodological approach for quantitative evaluation of dynamic changes of proteins and protein networks as well as the validation and utilization of biological markers. Towards this goal the PI and the users of the proposed instrument, have developed and established methodologies that employ stable isotope labeled peptides, proteomes and multiple reaction monitoring (MRM)/mass spectrometry (MS) workflows for the quantification of proteins in human samples and animal and cellular model systems. A TSQ Vantage unit (Thermo-Scientific) combined with an Eksigent Nano 2DLC, which has been operational in the TRL since September 2010, will remain until March 2013 on a lease supported by the University of Pennsylvania. We propose to acquire this remarkably productive unit. Chang-Gyu Hahn, M.D, Ph.D. as the P.I will assume overall responsibility for the equipment. Leadership will be equally divided between Chang-Gyu Hahn, M.D, Ph.D. and Ian Blair, Ph.D. Functioning as Co-P.I.'s. Dr. Hahn will focus on the operational aspects and Dr. Blair will provide scientific oversight.

? DESCRIPTION (provided by applicant): The N-methyl-D-aspartate (NMDA) receptor hypofunction hypothesis is one of the leading postulates for the pathophysiology of schizophrenia (SCZ) and is supported by numerous pharmacologic, behavioral and genetic studies. Nevertheless, we have little insight into specific alterations in NMDAR signaling and its mechanistic basis in SCZ patients. This is a critical knowledge gap, which has impeded further development of this hypothesis and limited our efforts to identify specific therapeutic interventions. (Preliminary Data) As direct evidence for altered NMDA receptor (NMDAR) signaling, we found decreased NMDA/Glycine induced tyrosine phosphorylation of NMDAR subunit 2 (GluN2) and reduced downstream signaling in the postmortem dorsal lateral prefrontal cortex (DLPFC) of SCZ cases. These changes are not associated with decreased NMDARs but with reduced activity of a cascade of kinases- Src kinase, protein kinase C and Pyk2- which in concert decrease GluN2 tyrosine phosphorylation. We found multiple molecular alterations in the DLPFC of SCZ cases; increased PSD-95, increased erbB4 activity, decreased dysbindin -1 and RPTPa, each of which can induce Src hypoactivity. (Hypotheses) We hypothesize that hypoactivity of Src in the NMDAR complex (Src-NR) reduces GluN tyrosine phosphorylation and is caused by altered protein interactions in a network of Src-NR-associated proteins ( the Src-NR interactome), which can be leveraged to modify behavioral phenotypes of NMDAR hypoactivity. (Approach) We propose a human-rodent translation strategy, by which we analyze disease related alterations in postmortem brains and examine their underlying mechanisms in rodent studies. Aim 1 will further examine postmortem brains of an elderly and mid-life SCZ cohorts to identify molecular alterations in the Src-NR interactome in SCZ, Aim 2 will determine the role of protein interactions in Src-NR hypoactivity and test rescue strategies in ex vivo preparations of rodent and human postmortem tissues and Aim 3 will determine SCZ related behavior and EEG phenotypes of Src-/- mice and test if Src enhancement can rescue such phenotypes in vivo.

Project Summary (Significance) Multiple lines of evidence have implicated mGluR5 signaling for the pathophysiology and treatment of schizophrenia; yet possible dysregulation of mGluR5 signaling and their pathophysiologic roles are presently unknown. Recently, we found direct evidence for reduced mGluR5 signaling in the postmortem dorsolateral prefrontal cortex (DLPFC) of patients with schizophrenia. mGluR5 is physically connected with NMDA receptor (GluN) complexes in the postsynaptic density (PSD) and these two pathways facilitate the activity of each other. (Preliminary Studies) We examined the agonist-induced activation of mGluR5 signaling in the postmortem dorsolateral prefrontal cortex (DLPFC) derived from 17 matched pairs of patients and controls. We found a striking reduction in three pathways downstream to mGluR5 receptor activation; namely, Gq/11 activation and recruitment of PI3K and scaffolding proteins compared to controls (P<0.01). This dysregulation was accompanied by alterations in phosphorylation of mGluR5, important for receptor desensitization, and in mGluR5 association with RGS4, Preso 1, norbin and tamalin, which are critical for surface expression and clustering. Importantly, we find evidence supporting disruption of reciprocal facilitation between mGluR5 and GluN signaling in schizophrenia and thus hypofunction of one can further compromise the other. (Scientific Premise) Schizophrenia is associated with mGluR5 signaling hypoactivity mediated by altered mGluR5 phosphorylation and protein ? protein interactions (PrPrIs) in the receptor complexes. mGluR5 hypofunction can contribute to GluN signaling hypoactivity and vice versa via disruption of reciprocal facilitation. PrPrIs in the interactome may be a point of convergence for etiologic factors. (Specific Aims) To further characterize mGluR5 hypoactivity and its interplay with GluN signaling in schizophrenia, we will examine molecular underpinnings for mGluR5 hypoactivity on GluN signaling and vice versa in the DLPFC of patients compared to controls. (Aim 1). Our preliminary data and recent studies point to PrPrIs as substrates upon which various molecular alterations converge and precipitate glutamatergic dysregulation. Combining discovery and quantitative proteomics, we will analyze PrPrI alterations in the mGluR5-GluN interactome in patients at the basal level and in response to receptor activation (Aim 2). PrPrI alterations in the interactome can be traced to their etiologic underpinnings at the genomic, transcriptomic and proteomic levels. Cutting edge systems biology algorithms permit us to leverage largest genomics data sets presently available and impute DLPFC transcriptomics data of the same number of subjects. Genetic variants, imputed transcriptomic results, along with quantitative PSD proteomics results will be projected onto the interactome using heat diffusion based algorithms (Aim 3). This will identify etiologically significant and potentially targetable subnetworks in the interactome. (Impact) We will establish mGluR5 hypofunction as integral to glutamatergic dysregulation in schizophrenia and identify etiologic underpinnings and potential points of intervention.