Pathway and biomarker discovery in a posttraumatic stress disorder mouse model

Posttraumatic stress disorder (PTSD), a prevalent psychiatric disorder, is caused by exposure to a traumatic event. Individuals diagnosed for PTSD not only experience significant functional impairments but also have higher rates of physical morbidity and mortality. Despite intense research efforts, the neurobiological pathways affecting fear circuit brain regions in PTSD remain obscure and most of the previous studies were limited to characterization of specific markers in periphery or defined brain regions. In my PhD study, I employed proteomics, metabolomics and transcriptomcis technologies interrogating a foot shock induced PTSD mouse model. In addition, I studied the effects of early intervention of chronic fluoxetine treatment. By in silico analyses, altered cellular pathways associated with PTSD were identified in stress-vulnerable brain regions, including prelimbic cortex (PrL), anterior cingulate cortex (ACC), basolateral amygdala (BLA), central nucleus of amygdala(CeA), nucleus accumbens (NAc) and CA1 of the dorsal hippocampus. With RNA sequencing, I compared the brain transcriptome between shocked and control mice, with and without fluoxetine treatment. Differentially expressed genes were identified and clustered, and I observed increased inflammation in ACC and decreased neurotransmitter signaling in both ACC and CA1. I applied in vivo 15N metabolic labeling combined with mass spectrometry to study alterations at proteome level in the brain. By integrating proteomics and metabolomics profiling analyses, I found decreased Citric Acid Cycle pathway in both NAc and ACC, and dysregulated cytoskeleton assembly and myelination pathways in BLA, CeA and CA1. In addition, chronic fluoxetine treatment 12 hours after foot shock prevented altered inflammatory gene expression in ACC, and Citric Acid Cycle in NAc and ACC, and ameliorated conditioned fear response in shocked mice. These results shed light on the role of immune response and energy metabolism in PTSD pathogenesis. Furthermore, I performed microdialysis in medial prefrontal cortex and hippocampus to measure the changes in extracellular norepinephrine and free corticosterone (CORT) in the shocked mouse and related them to PTSD-like symptoms, including hyperaroual and contextual fear response. I found that increased free CORT was related to immediate stress response, whereas norepinephrine level, in a brain region specific manner, predicted arousal and contextual fear response one month after trauma. I also applied metabolomics analysis to investigate molecular changes in prefrontal microdialysates of shocked mice. Citric Acid Cycle, Glyoxylate and Dicarboxylate metabolism and Alanine, Aspartate and Glutamate metabolism pathways were found to be involved in foot shock induced hyperarousal. Taken together, my study provides novel insights into PTSD pathogenesis and suggests potential therapeutic applications targeting dysregulated pathways

Pathway and biomarker discovery in a posttraumatic stress disorder mouse model

Posttraumatic stress disorder (PTSD), a prevalent psychiatric disorder, is caused by exposure to a traumatic event. Individuals diagnosed for PTSD not only experience significant functional impairments but also have higher rates of physical morbidity and mortality. Despite intense research efforts, the neurobiological pathways affecting fear circuit brain regions in PTSD remain obscure and most of the previous studies were limited to characterization of specific markers in periphery or defined brain regions. In my PhD study, I employed proteomics, metabolomics and transcriptomcis technologies interrogating a foot shock induced PTSD mouse model. In addition, I studied the effects of early intervention of chronic fluoxetine treatment. By in silico analyses, altered cellular pathways associated with PTSD were identified in stress-vulnerable brain regions, including prelimbic cortex (PrL), anterior cingulate cortex (ACC), basolateral amygdala (BLA), central nucleus of amygdala(CeA), nucleus accumbens (NAc) and CA1 of the dorsal hippocampus. With RNA sequencing, I compared the brain transcriptome between shocked and control mice, with and without fluoxetine treatment. Differentially expressed genes were identified and clustered, and I observed increased inflammation in ACC and decreased neurotransmitter signaling in both ACC and CA1. I applied in vivo 15N metabolic labeling combined with mass spectrometry to study alterations at proteome level in the brain. By integrating proteomics and metabolomics profiling analyses, I found decreased Citric Acid Cycle pathway in both NAc and ACC, and dysregulated cytoskeleton assembly and myelination pathways in BLA, CeA and CA1. In addition, chronic fluoxetine treatment 12 hours after foot shock prevented altered inflammatory gene expression in ACC, and Citric Acid Cycle in NAc and ACC, and ameliorated conditioned fear response in shocked mice. These results shed light on the role of immune response and energy metabolism in PTSD pathogenesis. Furthermore, I performed microdialysis in medial prefrontal cortex and hippocampus to measure the changes in extracellular norepinephrine and free corticosterone (CORT) in the shocked mouse and related them to PTSD-like symptoms, including hyperaroual and contextual fear response. I found that increased free CORT was related to diate stress response, whereas norepinephrine level, in a brain region specific manner, predicted arousal and contextual fear response one month after trauma. I also applied metabolomics analysis to investigate molecular changes in prefrontal microdialysates of shocked mice. Citric Acid Cycle, Glyoxylate and Dicarboxylate metabolism and Alanine, Aspartate and Glutamate metabolism pathways were found to be involved in foot shock induced hyperarousal. Taken together, my study provides novel insights into PTSD pathogenesis and suggests potential therapeutic applications targeting dysregulated pathways

Pathway and biomarker discovery in a posttraumatic stress disorder mouse model

Posttraumatic stress disorder (PTSD), a prevalent psychiatric disorder, is caused by exposure to a traumatic event. Individuals diagnosed for PTSD not only experience significant functional impairments but also have higher rates of physical morbidity and mortality. Despite intense research efforts, the neurobiological pathways affecting fear circuit brain regions in PTSD remain obscure and most of the previous studies were limited to characterization of specific markers in periphery or defined brain regions. In my PhD study, I employed proteomics, metabolomics and transcriptomcis technologies interrogating a foot shock induced PTSD mouse model. In addition, I studied the effects of early intervention of chronic fluoxetine treatment. By in silico analyses, altered cellular pathways associated with PTSD were identified in stress-vulnerable brain regions, including prelimbic cortex (PrL), anterior cingulate cortex (ACC), basolateral amygdala (BLA), central nucleus of amygdala(CeA), nucleus accumbens (NAc) and CA1 of the dorsal hippocampus. With RNA sequencing, I compared the brain transcriptome between shocked and control mice, with and without fluoxetine treatment. Differentially expressed genes were identified and clustered, and I observed increased inflammation in ACC and decreased neurotransmitter signaling in both ACC and CA1. I applied in vivo 15N metabolic labeling combined with mass spectrometry to study alterations at proteome level in the brain. By integrating proteomics and metabolomics profiling analyses, I found decreased Citric Acid Cycle pathway in both NAc and ACC, and dysregulated cytoskeleton assembly and myelination pathways in BLA, CeA and CA1. In addition, chronic fluoxetine treatment 12 hours after foot shock prevented altered inflammatory gene expression in ACC, and Citric Acid Cycle in NAc and ACC, and ameliorated conditioned fear response in shocked mice. These results shed light on the role of immune response and energy metabolism in PTSD pathogenesis. Furthermore, I performed microdialysis in medial prefrontal cortex and hippocampus to measure the changes in extracellular norepinephrine and free corticosterone (CORT) in the shocked mouse and related them to PTSD-like symptoms, including hyperaroual and contextual fear response. I found that increased free CORT was related to immediate stress response, whereas norepinephrine level, in a brain region specific manner, predicted arousal and contextual fear response one month after trauma. I also applied metabolomics analysis to investigate molecular changes in prefrontal microdialysates of shocked mice. Citric Acid Cycle, Glyoxylate and Dicarboxylate metabolism and Alanine, Aspartate and Glutamate metabolism pathways were found to be involved in foot shock induced hyperarousal. Taken together, my study provides novel insights into PTSD pathogenesis and suggests potential therapeutic applications targeting dysregulated pathways

Dynamic Patterns of Threat-Associated Gene Expression in the Amygdala and Blood

Stress and trauma profoundly influence psychiatric biobehavioral outcomes. The identification of treatment and biomarker targets would be accelerated by a broad understanding of the biological responses to these events. The goal of this study was to determine genes responsive to auditory fear conditioning (FC), a well-characterized amygdala-dependent rodent model of threat-exposure, in the presence or absence of prior stress history, providing insight into the physiological processes underlying response to trauma. RNA-sequencing was performed in blood and amygdala from mice that underwent fear conditioning with (Immo+FC) and without (FC) prior immobilization stress, a paradigm that induces HPA axis, and behavioral stress sensitization. In the amygdala, 607 genes were regulated by FC vs. home-cage (HC) controls, and 516 genes differed in stress-sensitized mice (Immo+FC vs. FC). In the former, we observed an enhancement of specific biological processes involved in learning and synaptic transmission, and in the latter processes associated with cell proliferation and the cellular response to drugs. In the blood of stress-sensitized animals, 468 genes were dynamically regulated when compared to FC, and were enriched for the biological pathways of inflammation and cytokine signaling. This study identified genes and pathways that respond to threat in the amygdala and blood of mice with and without a prior stress history and reveals the impact of stress history on subsequent inflammation. Future studies will be needed to examine the role of these dynamically regulated genes may play in human clinical stress and trauma-related disorders

Resilience to social defeat-induced increased in ethanol intake: neuroinflammation response

El estrés social es el principal factor de riesgo de las conductas adictivas. El fenotipo susceptible al estrés se ha convertido en la última década en un objetivo de estudio para la aplicación de tratamientos eficaces que mejoren la resiliencia al estrés. La promoción del afrontamiento activo mediante intervenciones farmacológicas y ambientales positivas está científicamente validada en el tratamiento de las adicciones. Sin embargo, estas intervenciones para promover la resiliencia durante la adolescencia como medida preventiva de experiencias estresantes intensas durante la edad adulta no han sido ampliamente estudiadas. Por ello, el objetivo principal de la presente Tesis Doctoral fue desarrollar estrategias farmacológicas y ambientales para potenciar la resiliencia ante los efectos negativos inducidos por la derrota social sobre el comportamiento y la respuesta neuroinflamatoria. Para alcanzar este objetivo, primero confirmamos y caracterizamos el potencial neuroinflamatorio del estrés por derrota social y su mediación en el aumento del consumo de etanol utilizando el paradigma operante de autoadministración oral. Posteriormente, basándonos en informes anteriores de nuestro laboratorio y de otros, evaluamos el potencial de la administración de oxitocina o del ejercicio físico para amortiguar los efectos negativos de la derrota social sobre el consumo de etanol y la respuesta neuroinflamatoria. Tras confirmar que estas intervenciones potencian la resiliencia a los efectos deletéreos de la derrota social, nos propusimos caracterizar el potencial preventivo del ejercicio, el enriquecimiento ambiental o la inoculación de estrés durante la adolescencia. Estos estudios caracterizaron además la vulnerabilidad y resiliencia a la experiencia de estrés social durante la adolescencia en ratones, ya que la respuesta a la derrota social es compleja y única durante esta etapa de la vida. Finalmente, se inició una nueva línea de investigación durante una estancia de investigación internacional para profundizar en la caracterización de los fenotipos resiliente/susceptible, centrándose en el estudio de los cambios morfológicos neuronales y sinápticos y la astrogliosis en estructuras cerebrales clave. Los resultados obtenidos en esta Tesis Doctoral nos permiten confirmar que la derrota social contribuye al desarrollo de conductas de evitación social y aumenta el consumo de etanol a largo plazo a través de la activación del sistema inmune. Hemos confirmado la existencia de fenotipos distintos basados en las respuestas conductuales de evitación social inducidas por la derrota social. El estrés social produce efectos negativos, pero no en todos los animales expuestos, ya que sólo un porcentaje de ellos desarrolla un perfil susceptible a comportamientos de tipo depresivo y a un mayor consumo de alcohol. Además, hemos caracterizado a los animales susceptibles y resilientes a los efectos de la derrota social, tanto cuando ésta se produce en la edad adulta como cuando se experimenta en la adolescencia. Pero nuestro principal objetivo ha sido potenciar la respuesta de resiliencia y hemos demostrado que intervenciones farmacológicas como la administración de oxitocina o ambientales como el ejercicio físico pueden potenciar la respuesta de resiliencia. Sin embargo, estas intervenciones también pueden aplicarse de forma preventiva durante la adolescencia. Por ejemplo, el ejercicio físico, el enriquecimiento ambiental o la exposición a un estresor social de baja intensidad antes de experimentar la derrota social inducen una potente respuesta resiliente. Estas intervenciones contribuirán al desarrollo de terapias preventivas y terapéuticas individualizadas en el tratamiento de los trastornos adictivos. Todos estos efectos protectores pueden ser producidos por diversos mecanismos, algunos de ellos estudiados en esta Tesis Doctoral, como los cambios en el BDNF o en la arquitectura neuronal. Sin embargo, el mecanismo fundamental que parece ser común en los animales resilientes es la disminución de la respuesta neuroinflamatoria que siempre se observa aumentada en los animales susceptibles a la derrota.Social stress is the main risk factor for addictive behaviors. Stress-susceptible phenotypes have become in the last decade a target of study for the application of effective treatments to enhance stress resilience. Promoting active coping through positive pharmacological and environmental interventions is scientifically validated in the treatment of addictions. However, these interventions to promote resilience during adolescence as a preventive measure for intense stressful experiences during adulthood have not been widely studied. Therefore, the main aim of the present Doctoral Thesis was to develop pharmacologic and environmental strategies to enhance resilience to the negative effects induced by social defeat on behavior and the neuroinflammatory response. To reach this aim, we first confirmed and characterized the neuroinflammatory potential of social defeat stress and its mediation of increased ethanol consumption using the operant paradigm of oral self-administration. Subsequently, based on previous reports from our laboratory and others, we assessed the potential of oxytocin administration or physical exercise to buffer the negative effects of social defeat on ethanol consumption and the neuroinflammatory response. After confirming that these interventions potentiate resilience to the deleterious effects of social defeat, we set out to characterize the preventive potential of exercise, environmental enrichment or stress inoculation during adolescence. These studies further characterized the vulnerability and resilience to social stress experience during adolescence in mice, since the response to social defeat is complex and unique during this stage of life. Finally, a new line of research was initiated during an international research stay to further characterize resilient/susceptible phenotypes, focusing on the study of neuronal and synaptic morphological changes and astrogliosis in key brain structures. The results obtained in this Doctoral Thesis allow us to confirm that social defeat contributes to the development of social avoidance behaviors and increases long-term ethanol consumption through the activation of the immune system. We have confirmed the existence of distinct phenotypes based on social avoidance behavioral responses induced by social defeat. Social stress produces negative effects, but not in all exposed animals, since only a percentage of them develop a profile susceptible to depressive-type behaviors and increased alcohol consumption. Moreover, we have characterized susceptible and resilient animals to the effects of social defeat, both when it occurs in adulthood and when it is experienced in adolescence. But our main goal has been to enhance the resilience response and we have shown that pharmacological interventions such as oxytocin administration or environmental interventions such as physical exercise can enhance the resilience response. However, these interventions can also be applied preventively during adolescence. For example, physical exercise, environmental enrichment or exposure to a low-intensity social stressor before experiencing social defeat induce a powerful resilient response. These interventions will contribute to the development of individualized preventive and therapeutic therapies in the treatment of addictive disorders. All these protective effects can be produced by various mechanisms, some of them studied in this Doctoral Thesis, such as changes in BDNF or in neuronal architecture. However, the fundamental mechanism that appears to be common in resilient animals is a decrease in the neuroinflammatory response that is always observed to be increased in susceptible defeated animals

THE RELATIONSHIP BETWEEN ATTACHMENT AND SERUM OXYTOCIN AND HEAT SHOCK PROTEIN-70 LEVELS IN ADOLESCENTS OF PARENTS WITH SCHIZOPHRENIA AND BIPOLAR DISORDER

Background: The aim of this study was to evaluate serum heat shock protein 70 (HSP70) and oxytocin levels, attachment and perceived social support levels in adolescents with parental bipolar disorder (BD) and Schizophrenia (SCZ). Subjects and Methods: This study included 9 adolescents with SCZ parents, 30 adolescents with BD parents and 31 healthy adolescents. Brief Symptom Inventory (BSI), Relationship Scale Questionnaire-Adolescent Form (RSQ-A) and Multidimensional Scale of Perceived Social Support (MSPSS) were administered to all participants. In addition, serum HSP-70 and oxytocin levels were evaluated. Results: There was no significant difference between the groups in terms of attachment style, psychiatric symptoms and perceived social support. Serum HSP-70 levels were found to be lower in adolescents whose parents had BD. Serum oxytocin levels of the SCZ group were significantly lower than those of the BD group. Conclusions: HSP-70 level was found to be lower in adolescents with BD parents. Oxytocin level was found to be lower in adolescents with SCZ parents. These findings suggest that HSP-70 and oxytocin may be a marker of early life stress in adolescents with parental psychopathology. However, studies are needed to evaluate the relationship between attachment, oxytocin and HSP-70 in adolescents exposed to parental psychopathology in early life

Activation of the pro-resolving receptor Fpr2 attenuates inflammatory microglial activation

Poster number: P-T099 Theme: Neurodegenerative disorders & ageing Activation of the pro-resolving receptor Fpr2 reverses inflammatory microglial activation Authors: Edward S Wickstead - Life Science & Technology University of Westminster/Queen Mary University of London Inflammation is a major contributor to many neurodegenerative disease (Heneka et al. 2015). Microglia, as the resident immune cells of the brain and spinal cord, provide the first line of immunological defence, but can become deleterious when chronically activated, triggering extensive neuronal damage (Cunningham, 2013). Dampening or even reversing this activation may provide neuronal protection against chronic inflammatory damage. The aim of this study was to determine whether lipopolysaccharide (LPS)-induced inflammation could be abrogated through activation of the receptor Fpr2, known to play an important role in peripheral inflammatory resolution. Immortalised murine microglia (BV2 cell line) were stimulated with LPS (50ng/ml) for 1 hour prior to the treatment with one of two Fpr2 ligands, either Cpd43 or Quin-C1 (both 100nM), and production of nitric oxide (NO), tumour necrosis factor alpha (TNFα) and interleukin-10 (IL-10) were monitored after 24h and 48h. Treatment with either Fpr2 ligand significantly suppressed LPS-induced production of NO or TNFα after both 24h and 48h exposure, moreover Fpr2 ligand treatment significantly enhanced production of IL-10 48h post-LPS treatment. As we have previously shown Fpr2 to be coupled to a number of intracellular signaling pathways (Cooray et al. 2013), we investigated potential signaling responses. Western blot analysis revealed no activation of ERK1/2, but identified a rapid and potent activation of p38 MAP kinase in BV2 microglia following stimulation with Fpr2 ligands. Together, these data indicate the possibility of exploiting immunomodulatory strategies for the treatment of neurological diseases, and highlight in particular the important potential of resolution mechanisms as novel therapeutic targets in neuroinflammation. References Cooray SN et al. (2013). Proc Natl Acad Sci U S A 110: 18232-7. Cunningham C (2013). Glia 61: 71-90. Heneka MT et al. (2015). Lancet Neurol 14: 388-40