Etienne Sibille - US grants

[unreadable] DESCRIPTION (provided by applicant): Major depression is emerging as a disease of the central nervous system with a characteristic pathophysiology. For inpatients with major depression, the lifetime mortality due to suicide reaches 15%. Despite a global burden of neuropsychiatric disorders on individual mental health and society's productivity, little is known on mechanisms or molecules that are responsible for these diseases. Accordingly, the identification of the neurobiological underpinnings for mood disorders represents an important challenge in neuropsychiatric research. Complex polygenic mechanisms are likely, with environmental factors contributing to the development of depression and suicidal behavior. However, research efforts have been largely limited to components of brain biology that are affected by antidepressants and mood stabilizers. Microarray technology and genomic characterization represent new investigational developments that allow for unbiased identification of new molecular components and mechanisms of mood disorders. During the award period, the applicant aims to train in the application of microarrays to postmortem human brain studies, to initiate a genomic and molecular characterization of prefrontal cortex dysfunction in depressed patients who died by suicide, and to seek new mechanisms, genes and molecules that contribute to the development of the disease. Preliminary results indicate a rich potential for genomic approaches to psychiatric disorders and hints at a possible molecular classification of depression into unrecognized subtypes. The specific aims of the study are /) to characterize gene expression profiles in the prefrontal cortex of human subjects, ii) to assess the effect of major depression and suicide on gene expression, iii) to investigate genes and molecular pathways that were affected by the disease, and iv) to develop new understanding of biological mechanisms involved in depression and suicide, with possibilities for designing new genetic mouse models, and uncovering new molecular targets for treatment and prevention of mood disorders. The proposed training plan will enable the applicant to benefit from recent advances in genome sequencing and genomic technologies, and to develop expertise to bridge these approaches to the study of neuropsychiatric disorders. In particular, the applicant will receive valuable training in genomics, statistics and bioinformatics, and in clinical and basic science aspects of brain function. The training program proposes formal courses, supervised training and frequent interactions with several research groups involved in pioneering aspects of genomic research in mood disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Despite the substantial personal and economic burden of mood disorders, understanding the pathological and molecular features of these disorders remains a considerable challenge in psychiatric research. Dysregulated serotonergic and stress pathways appear to be contributing factors in major depressive disorder (MOD);however, it is likely that numerous other unidentified risk factors exist. Here we propose to investigate the molecular pathology of MOD, using a combined approach of microarray experiments, bioinformatic analysis and anatomical characterization of results. Our central hypothesis states that the biological liability to MOD is reflected in a persistent molecular pathology that is detectable in the postmortem human brain and that affects a cortico-limbic network, whose dysfunction might specifically cause, or at least correlate with, the affective component of MOD. Hence, based on microanatomical and functional studies, we will concentrate on two densely interconnected brain areas within this cortical-limbic network of MOD: i) the amygdala (AMY), as a brain region that is crucial to the integration and expression of emotions, and ii) the anterior cingulate cortex (ACC), as MDD-related functional and morphological changes have been consistently reported in this brain area. Importantly, no gene expression profiling studies have been reported in the AMY, and by extension no comparative analysis of potential molecular correlates of MOD have been reported between the AMY and ACC, especially in the context of neuropsychiatric disorders. As microanatomical studies suggest a glial MDD-related pathology in these two brain areas, we will apply novel analytical approaches to separately assess the contribution of altered glial or neuronal functions within the gray matter in correlation with MDD. Results from this research proposal could reveal either a general pathway that is common among all MDD subjects and/or specific pathways that may differ as a function of sex and family history of MDD, two factors that are associated with different phenotypic features of MMD. Results will also determine the potential contribution of several disease factors to the transcriptome correlates of MDD, including death by suicide, co-morbid alcohol dependence or antidepressant treatment. The characterization of patterns of nuclei (AMY) and laminar (ACC) changes for selected genes will provide anatomical information to generate network-based hypotheses on the molecular pathology of MDD.

DESCRIPTION (provided by applicant): Although depression is a leading cause of disability worldwide and clinically-effective antidepressants were discovered over 50 years ago, the cellular and molecular pathology of depression remains mostly uncharacterized. As a result, very few new drug classes have emerged and a large proportion of depressed subjects do not get adequate relief. Yet, this is surprising since altered mood regulation, the core feature of depression, is subserved by identified neural networks including the amygdala, is precipitated by environmental factors such as chronic stress, includes neuroendocrine dysfunctions, and is modulated by known risk factors (female sex, altered serotonin transporter [SERT] and prior depressive episodes). Thus, to develop a plausible animal model that recapitulates these known features of the illness would be invaluable in facilitating the identification of relevant biological and molecular disturbances in depression. Unpredictable chronic mild stress (UCMS) in rodents mimics in a naturalistic way both the role of psycho- social stress in eliciting emotion-related behavioral changes that are reminiscent of symptoms of depression, and the timeframe of therapeutic response to antidepressant treatment. We now show that UCMS in mice also recapitulates the increased vulnerability to altered mood states associated with female sex or low SERT, and that UCMS induces changes in gene expression in the amygdala that are phylogenetically conserved with the molecular signature of human depression. Thus, with the long-term goal of investigating causal mechanisms of depression, this proposal will first characterize UCMS as an integrated rodent model of human depression by assessing the contribution of known risk factors (female sex, low SERT and prior depressive episodes) to the behavioral (Aim 1), neuroendocrine (Aim 2) and molecular (Aim 3) correlates of UCMS. Second, this model will be used, in combination with our identified biosignature of depression in the human amygdala, to identify shared downstream biological pathways that may support the pathophysiology of depression. Consistent with NIMH goals, this R21 proposal investigates an integrated rodent model of depression to better understand the contribution of genetic, neurobiological, behavioral and environmental factors to the development and trajectory of mental disorders. Importantly, this proposal also benefits from a research team actively investigating human postmortem pathology. For instance, our identification of a molecular signature of human depression that is conserved in rodent UCMS will allow direct validation of the rodent molecular findings at modeling mechanisms that are relevant to human depression. Specifically, a biological feature that is i) temporally correlated with changes in emotion-related behaviors in mice, ii) modulated by risk factors known to affect vulnerability to depression in humans, and iii) supported by equivalent molecular changes as in human depression, will represent first a robust and valid candidate system for future investigation of mechanisms leading to altered mood states in depression, and, second, a potential new drug target. PUBLIC HEALTH RELEVANCE: This research proposal investigates an integrated rodent model of depression in order to better understand the contribution of genetic, neurobiological, behavioral and environmental factors to the development and trajectory of the human illness. Specifically, we will investigate biological events that are temporally correlated with changes in emotion-related behaviors in mice, modulated by risk factors known to affect vulnerability to depression in humans, and supported by equivalent molecular changes as in human depression. Results from these studies will identify first robust and valid candidate neurobiological brain systems for future investigations of mechanisms leading to depression, and, second, potential new drug targets.

DESCRIPTION (provided by applicant): Despite the substantial personal and economic burden of mood disorders, understanding the pathological and molecular features of these disorders remains a considerable challenge in psychiatric research. Dysregulated serotonergic and stress pathways appear to be contributing factors in major depression; however, it is likely that numerous other unidentified risk factors exist. Here we propose to investigate the molecular pathology of major depression, using a combined approach of microarray experiments, bioinformatic analysis and anatomical characterization of results. Our central hypothesis states that the biological liability to major depression is reflected in a persistent molecular pathology that is detectable in the postmortem human brain and that affects a cortico-limbic network, whose dysfunction might specifically cause, or at least correlate with, the affective component of depression. Hence, based on microanatomical and functional studies, we will concentrate on two densely interconnected brain areas within this cortical- limbic network of mood regulation: i) the amygdala (AMY), as a brain region that is crucial to the integration and expression of emotions, and ii) the anterior cingulate cortex (ACC), as depression-related functional and morphological changes have been consistently reported in this brain area. As microanatomical studies suggest a glial depression-related pathology in these two brain areas, we will apply novel analytical approaches to separately assess the contribution of altered glial or neuronal functions within the gray matter in correlation with major depression. Together, results from this research proposal could reveal either a general pathway that is common among all depressed subjects and/or specific pathways that may differ as a function of sex and family history of major depression, two factors that are associated with different phenotypic features of depression. The characterization of patterns of nuclei (AMY) and laminar (ACC) changes for selected genes will provide anatomical information to generate network-based hypotheses on the molecular pathology of depression.

DESCRIPTION (provided by applicant): Altered emotion regulation and low mood states in major depressive disorder (MDD) are thought to reflect altered corticolimbic structure and function in affected individuals, and involve the dorsolateral prefrontal cortex (DLPFC), the subgenual anterior cingulate cortex (sgACC) and the amygdala. Specifically, low GABA-related inhibition may underlie inadequate sensing and relaying of emotionally-salient stimuli by the amygdala and deficient integration of this relayed information by the sgACC with higher DLPFC processing. However, primary cellular and molecular evidence for these proposals are sparse. We have now identified a selective downregulation of somatostatin (SST), an inhibitory neuropeptide expressed in a subset of GABA neurons, in the DLPFC, sgACC and amygdala of subjects with MDD. Low SST was confirmed across cohorts, affected female subjects more robustly, and moderately correlated with the severity of the illness. At the microcircuitry level, SST GABA neurons target the distal dendrites of pyramidal neurons, but different subsets of SST neurons display different anatomical localizations and physiological properties, potentially mediating different information processing. Together, this lead us to hypothesize that low SST levels in specific cortical layers and amygdala nuclei are critical risk factors for MDD, and that additional factors determine the severity of the illness. Accordingly, we further hypothesize that, under challenging conditions, suboptimal SST function may trigger cellular and molecular maladaptive changes that lead to altered emotion regulation and low mood states, changes that will provide leads for the future development of novel therapeutics. Hence, to investigate the contribution of SST in mood regulation, we will first characterize the anatomical and microcircuitry specificity of changes in the human postmortem brains of MDD subjects (cortical layer & amygdala nuclei) (Aim 1). We will then test the low SST - risk factor hypothesis in a rodent model, and predict that mice with low SST (heterozygous mutant) will display increased vulnerability to develop high anxiety-like/depressive-like behaviors (defined as emotionality) after unpredictable chronic mild stress (Aim 2). Finally, we will use mice expressing green fluorescent protein in the SST interneuron subtype(s), which most closely corresponds to the subtype affected in MDD, to investigate low SST- and stress-associated cell-specific maladaptive molecular changes (Aim 3). In summary, low SST in MDD provide a cellular and molecular lead to investigate mechanisms of the illness, and a potential molecular link between foremost hypotheses for biological disturbances in MDD (neural network dysfunction and reduced GABA-related function). However, SST also assumes several central and peripheral functions, and pharmacological manipulation has multiple (side-)effects, so it is essential to identify maladaptive molecular changes in SST GABA neurons, as potential mediators of the phenotype and as novel and potentially selective targets for the future investigation and development of novel therapeutics aimed at relieving the burden of MDD.

DESCRIPTION (provided by applicant): Improvements in medical interventions and living conditions have dramatically increased the average human lifespan over the last century. As a result, emotional and cognitive fitness has become a major determinant, and unmet challenge, to the quality of life during old age. Indeed, if successful aging is achievable, for numerous individuals, low mood is too often an early symptom and significant contributor to the downward spiral of aging, which includes further cognitive and motor declines. Low mood is also observed in presymptomatic stages of neurodegenerative disorders, together suggesting that mood regulation may be selectively vulnerable at the vigor-to-frailty transition. However, this hypothesis has been difficult to test, as correlations between molecular changes and functional declines are logistically complex to assess in elderly individuals, and, since means to identify individuals at higher biological risk are not available. Here, we propose to identify genetic modulators that associate with both biological and functional aging, by performing parallel postmortem and in vivo studies. Our assay for biological aging is based on the fact that brain aging associates with robust molecular, cellular and structural changes, for which we have identified a specific set of genes with age-dependent expression changes. This molecular signature of aging contains many neuropsychiatric and neurodegenerative disease-related genes, which are affected in disease-promoting directions, suggesting that aging may promote aspects of diseases. Notably, we show that molecular age - defined herein as the age that is predicted by the gene expression profile in that individual - can deviate from chronological age. For instance, individuals carrying a DNA variant in a putative longevity gene (Sirtuin 5) display older molecular ages, potentially through accelerated age-dependent declines in mitochondrial-related gene transcripts. Thus, we hypothesize that subjects carrying risk alleles associated with older brain molecular age will display higher incidence of mood and other low function symptoms, and conversely that subjects carrying protective alleles may experience greater successful aging. To test this hypothesis, we will systematically identify genetic variants associated with older or younger brain molecular ages in postmortem individuals (n~300) (Aim 1). We will then assess the extent to which the presence of these identified risk or protective genetic factors predicts corresponding functional outcomes in vivo in elderly individuals, as manifested by mood, cognitive and motor age-dependent changes in subjects from two epidemiological studies of aging: the Cardiovascular Health (n=5,888) and Health Aging and Body Composition (n=3,075) studies (Aim 2). At the end of this study, we will have achieved (i) the identification of robust genetic markers for biological and functional aging, and (ii) a more detailed characterization of biological/functional links between mood, cognition and motor functions. This will provide (iii) evidence-based hypotheses to monitor critical functional domains in old age, and (iv) biological leads for rational experimental design to investigate successful aging.

Project Summary / Abstract Personalized medicine will require the use of discriminative tests that are predictive of disease state and individual treatment response. Major depressive disorder (MDD) is a heterogeneous illness that is the leading cause of disability in developed countries and a major cause of premature death due to suicide. Research aimed at characterizing the pathology of MDD however, has mostly focused on central mechanisms and has only occasionally been translated into hypotheses that can be tested in clinical settings using readily available peripheral blood samples from human subjects (and with modest results for neuroendocrine tests for instance). Focusing on altered transcriptome in relevant brain areas, we have now identified gene expression changes that are associated with ¿depressed¿ states in both the human postmortem brain and the unpredictable chronic mild stress (UCMS) rodent model of depression, and that are reversed by antidepressant (AD) treatments in mice. The presence of this conserved ¿biosignature of MDD¿ demonstrates that MDD is associated with persistent molecular pathologies, and that a parallel study in a more tractable animal model of the illness can support the analysis of the complex human data in identifying conserved and state-dependent disease-related changes. How can we translate these findings into a potential clinical tool? The identification of peripheral biomarkers co-varying with disease states and treatment response is a primary goal in developing a biological (in addition to symptomatic) definition of MDD. Studies showed that blood transcriptomes largely parallel central profiles and can provide valuable information on subject-specific parameters, including for neuropsychiatric disorders. Hence, based on our prior findings in the brain, we hypothesize that MDD-related blood changes will manifest as conserved gene changes, for which the UCMS rodent model will provide an independent line of validation and a means to assess confounding effects of AD treatments, together facilitating the identification of MDD- and relevant gene transcript changes as putative peripheral disease biomarkers. To optimize our probability of success, we will assess patients with MDD and co-occurring anxiety receiving citalopram treatment and psychotherapy (to augment AD compliance). Blood transcriptome changes will be assessed in patients (n=40) before and after AD treatment and in control subjects (n=20) (Aim 1) to investigate the potential of peripheral gene changes to track disease state and treatment response/non-response. Parallel studies in UCMS-exposed and AD-treated mice will help us distinguish candidate biomarkers from confounding AD effects in human subjects, and will begin assessing brain-blood conservation of changes (Aim 2). Follow-up studies will assess the sensitivity and disease specificity of the identified biomarkers, extend findings from the response to the remission timeframe, and will lead to biological predictions about disease state, risk factors and treatment responses, which will be tested in future studies in larger clinical cohorts.

DESCRIPTION (provided by applicant): Major depressive disorder (MDD) is a heterogeneous illness with many clinical variables - such as sex, age, alcohol, antidepressant drug, recurrence or death by suicide -- as potential factors characterizing subtypes of MDD, making it difficult to fully understand its underlying mechanisms and heterogeneous genetic underpinnings. Many transcriptomic studies have been generated in the literature, including those from Dr. Sibille, the co-PI on this proposal. We propose to apply state-of-the-art statistical integrative analyses tailored to combine multiple MDD transcriptomic studies that will address the specific issues of case-control pairing design, confounding clinical variables and small sample size. Our study will detect novel MDD associated biomarkers, pathways and co-expression modules, and elucidate the magnitudes of transcriptomic changes attributable to substance abuse, recurrence, disease severity, age and sex. The results will enhance our understanding to MDD genetic mechanisms and lead to better and individualized therapeutic solutions. PUBLIC HEALTH RELEVANCE: Our proposed research is to develop and apply modern genomic meta-analysis methods to combine multiple major depressive disorder (MDD) transcriptomic studies that will adequately model the specific data structure of case-control pairing design, confounding clinical variables and small sample size. The goal is to detect novel MDD associated biomarkers, pathways and co-expression modules, and elucidate the magnitudes of transcriptomic changes attributable to substance abuse, recurrence, disease severity, age and sex. The results will enhance our understanding of MDD genetic mechanisms and lead to better and individualized therapeutic solutions.

DESCRIPTION (provided by applicant): Major depressive disorder (MDD) is a heterogeneous illness with many clinical variables - such as sex, age, alcohol, antidepressant drug, recurrence or death by suicide -- as potential factors characterizing subtypes of MDD, making it difficult to fully understand its underlying mechanisms and heterogeneous genetic underpinnings. Many transcriptomic studies have been generated in the literature, including those from Dr. Sibille, the co-PI on this proposal. We propose to apply state-of-the-art statistical integrative analyses tailored to combine multiple MDD transcriptomic studies that will address the specific issues of case-control pairing design, confounding clinical variables and small sample size. Our study will detect novel MDD associated biomarkers, pathways and co-expression modules, and elucidate the magnitudes of transcriptomic changes attributable to substance abuse, recurrence, disease severity, age and sex. The results will enhance our understanding to MDD genetic mechanisms and lead to better and individualized therapeutic solutions.

Project 1 (P1) provides molecular tests at the cell type-specific level for the following aspects of the Central Hypothesis: 1) a core cortical pathology in schizophrenia affects layer 3 pyramidal cells, and 2) the impact of this pathology on gene expression is moderated by factors that differ across cortical regions. Layer 3 pyramidal cells will be collected using laser microdissection in postmortem samples from subjects with schizophrenia or schizoaffective disorder and healthy comparison subjects (Aim 1). Within-region analyses will inform on the cell autonomous pathology of layer 3 pyramidal cells in schizophrenia. Across-region analyses will characterize molecular regional specificities in control subjects and how they impact the pathology of layer 3 pyramidal cells in schizophrenia. A secondary goal is to gain insight into potential molecular targets to restore functional balance along the visual working memory and attention network. Accordingly, since layer 3 pyramidal cell alterations are predicted to induce transcriptional responses in the parvalbumin (PV)-containing GABA neurons that they innervate, similar analyses will be performed in PV neurons (Aim 2). To provide a comprehensive molecular perspective on coordinated local circuit alterations, expression of genes (mRNA) and gene regulatory micro-RNAs (miRNA) will be investigated in parallel (Aims 1-2) and biological modules implicated in healthy control and pathological conditions will be identified by changes in coordinated expression across cell types, regions and cohorts (Aim 3). Results from PI will inform all other Center projects on the molecular and cellular bases in regards to the Central Hypothesis that regional differences moderate the serverity of layer 3 PC alterations in schizophrenia. PI will also provide molecular leads for protein marker studies in P2, and for layer 3 PC regional differences in electrophysiological properties in P3. Together Projects 1-3 will provide the molecular, cellular and circuitry bases for the functional connectivity studies in monkey (P4), and in imaging studies of healthy controls and subjects with schizophrenia (P5).