Chiaki Fukuhara - US grants

Our long-term research objective is to understand the molecular mechanisms that initiate circadian gene expression following external stimulation. We will use the pineal gland to study signaling pathways because it has been extensively used to study signaling cascades and so provides an extensive knowledgebase for our studies. Our research proposal has two goals: (1) to understand the mechanisms by which norepinepherine (NE) controls Period1 expression and (2) to investigate the regulation of circadian "Clock" genes' expressions following external stimulation. First, since NE increases intra-cellular cyclic AMP (cAMP) and cyclic GMP (cGMP) levels, we will investigate the mechanisms by which cAMP and cGMP signaling pathways induce Period 1 mRNA levels. Since Period 1 is thought to be involved in the resetting of circadian rhythms, we will focus on the regulation of Period 1 expression in specific aims I and I1. Secondly, we will investigate whether or not other circadian "Clock" mRNA levels are induced by NE, cAMP, cGMP, or cAMP and cGMP co-stimulation. Besides its biological importance and interest, the use of pineal gland as a model, has promise for providing information that may be applicable to the prevention of neurodegenerative diseases, such as Alzheimer's disease, since it has been suggested that melatonin may play a role in neuroprotection. In addition, our studies have the pete" tial to address problems associated with jet lag, circadian-based sleep disorders, some neuropsychiatric illnesses, and shift work.

Advanced Sleep Phase Syndrome; Advanced Sleep Phase Syndrome (ASPS); Affective Psychosis, Bipolar; Assay; Bioassay; Biologic Assays; Biological Assay; Biopsy; Bipolar Disorder; Blood; Blood Cells; Blood Sample; Blood specimen; CRISP; Cancers; Cells; Chemicals; Cicatrix; Circadian Rhythms; Clinical; Computer Retrieval of Information on Scientific Projects Database; Delayed Sleep Phase Syndrome; Dermal; Diagnostic; Disease; Disorder; Disruption; Diurnal Rhythm; Fibroblasts; Funding; Future; Generations; Genes, Reporter; Genetic; Grant; Health; Human; Human, General; In Vitro; Institution; Invasive; Investigators; Left; Link; Malignant Neoplasms; Malignant Tumor; Man (Taxonomy); Man, Modern; Measurement; Measures; Mental disorders; Mental health disorders; Metabolic syndrome; Methods and Techniques; Methods, Other; Monitor; NIH; National Institutes of Health; National Institutes of Health (U.S.); Numbers; Nyctohemeral Rhythm; Pain; Painful; Participant; Pathogenesis; Patients; Pattern; Peripheral; Peripheral Blood Cell; Pharmaceutical Agent; Pharmaceuticals; Pharmacologic Substance; Pharmacological Substance; Physiologic; Physiological; Procedures; Psychiatric Disease; Psychiatric Disorder; Psychosis, Manic-Depressive; Purpose; Recruitment Activity; Regulation; Reporter Genes; Reporting; Research; Research Personnel; Research Resources; Researchers; Resources; Reticuloendothelial System, Blood; Role; Scars; Seasonal Affective Disorder; Seasonal Mood Disorder; Skin; Sleep Disorders; Source; System; System, LOINC Axis 4; Techniques; Testing; Time; Twenty-Four Hour Rhythm; United States National Institutes of Health; Unspecified Mental Disorder; bipolar affective disorder; cell type; circadian; circadian process; daily biorhythm; disease/disorder; diurnal variation; healthy volunteer; in vitro Assay; macrophage; malignancy; manic depressive disorder; manic depressive illness; mental illness; neoplasm/cancer; psychological disorder; recruit; seasonal depression; sleep problem; social role; tool

Adverse effects; Affect; Affective Psychosis, Bipolar; Assay; Bioassay; Biologic Assays; Biological Assay; Biology; Bioluminescence; Bipolar Disorder; Blood Cells; Blood Serum; Cells; Cessation of life; Circadian Rhythms; Clinical; Comment; Comment (PT); Comment [Publication Type]; Commentary; Commentary (PT); Darkness; Darknesses; Data; Death; Depression; Dermatology; Development; Diagnostic; Disease; Disorder; Diurnal Rhythm; Drugs; E-Mail; Editorial Comment; Editorial Comment (PT); Effectiveness; Electronic Mail; Email; Emotional well being; Feels well; Fibroblasts; Future; Generations; Genes; Genetic Screening; Genome; Goals; High Throughput Assay; Human; Human, General; Immunologic, Luciferase; In Vitro; Li+ element; Lithium; Luciferases; Man (Taxonomy); Man, Modern; Manias; Manic; Manic State; Medical; Medication; Mental Depression; Mental disorders; Mental health disorders; Mental well-being; Methods; Modeling; Monitor; Mood stabilizers; Moods; Normal mental condition; Normal mental state; Normal psyche; Nyctohemeral Rhythm; Outcomes Research; Output; Pathogenesis; Patients; Peripheral; Peripheral Blood Cell; Personal Satisfaction; Pharmaceutic Preparations; Pharmaceutical Preparations; Pharmacology; Physiologic; Physiological; Population; Predisposition; Programs (PT); Programs [Publication Type]; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Proteins; Psychiatric Disease; Psychiatric Disorder; Psychological Well Being; Psychosis, Manic-Depressive; Published Comment; Purpose; Range; Recurrence; Recurrent; Regulation; Reporter; Reporting; Research; Research, Outcomes; Resistance; SCHED; Schedule; Schools, Medical; Screening procedure; Sense of well-being; Serum; Sleep; Suicide; Susceptibility; System; System, LOINC Axis 4; Testing; Therapeutic; Therapeutic Effect; Time; Treatment Side Effects; Twenty-Four Hour Rhythm; Universities; Unspecified Mental Disorder; Variant; Variation; Viewpoint; Viewpoint (PT); WHO; Well in self; World Health Organization; bipolar affective disorder; circadian; circadian clock; circadian pacemaker; circadian process; daily biorhythm; disability; disease/disorder; diurnal variation; drug/agent; fatal attempt; fatal suicide; gene product; high throughput screening; intent to die; manic depressive disorder; manic depressive illness; medical schools; mental illness; prevent; preventing; professor; programs; psychological disorder; psychological wellness; resistant; response; screening; screenings; self wellness; side effect; suicidality; therapy adverse effect; treatment adverse effect; well-being; years of life lost

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Disturbances in circadian rhythm expression appear to be associated with many diseases, such as seasonal affective disorders, a variety of other mental illnesses, sleep problems, and various metabolic syndromes. Even though associations between circadian rhythm disturbances and several diseases have been reported, the precise mechanisms by which circadian rhythm expression impacts the expression of diseases remain unknown. Among the limiting factors are technical difficulties related to the measurement of human circadian rhythm expression using physiological measures. To better understand human circadian rhythm expression in those patients and the implications of circadian rhythm disturbances in pathogenesis, as well as to evaluate potential pharmaceutical treatments, it is important to develop a non-invasive, simple, rapid and reliable in vitro assay to determine circadian rhythm expression in humans. One recently-developed technique allows us to obtain high-quality circadian rhythm records in real time, using the circadian clock-luciferase reporter gene and a lentivirus. Furthermore, researchers have demonstrated the feasibility of using skin fibroblasts to record human circadian rhythm expression. However, skin biopsy is not a routine procedure in most clinical departments, and they are painful for patients. Therefore, it would be beneficial to develop an assay system using some other cell types. Our preliminary data suggest that macrophages may be useful in this regard. In the present proposal, we aim to develop a non-invasive, in vitro assay system to measure human circadian rhythm in macrophages derived from human blood samples, using real-time monitoring of circadian parameters with a clock gene reporter (Aim 1). In addition, this technique will be applied to evaluate the effects of chemicals, thereby testing the feasibility of this assay (Aim 2) are confident to establish such an assay system using human blood cells, and determine the effects of various chemicals on circadian rhythm expression.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Bipolar disorder is characterized by recurrent episodes of mania and depression, a common psychiatric disorder. This disorder affects about 2.3 million adults every year in the US, costing $44 billion annually, but less than half of these patients are treated successfully. The mood stabilizer, lithium, has proven its efficacy in preventing the recurrence of mania and suicide more than any other drug for more than 50 years. In the past decade, however, lithium use in the United States has declined substantially, a trend not seen in the rest of the world. One of the reasons for this is the perception that lithium is more difficult to use. Additionally, only a subpopulation of patients shows benefits from lithium treatment, and therapeutic effects may not be fully apparent for several months. Therefore, it would be beneficial if there were a simple, rapid, and reliable assay that can be applied prior to treating patients with the drug to predict which patients will show a therapeutic response to lithium. Bipolar disorder patients with a family history of the disorder respond to lithium treatment better than those without such a family history, suggesting that lithium sensitivity is controlled in part by genetic factors. Since gene mutations affect not only the cells of the brain but also peripheral cells, lithium sensitivity may be predicted using peripheral cells. In the past, numerous studies have been conducted on gene expression by lithium stimulation in rodents and humans. The observation that lithium affects gene expression patterns in a variety of cells suggests that monitoring gene expression in human peripheral cells may provide a comprehensive diagnostic for patients'responsiveness to lithium. However, due to the lack of any animal or cell model regarding lithium sensitivity, no study exists in which differential gene expression is reported between lithium responders and nonresponders. Therefore, the goal of our present proposal is to identify a set of marker genes that are differentially expressed between lithium : responders and non-responders, and test our hypothesis that clinical response to lithium can be predicted using gene expression patterns in human peripheral cells. In addition to the P.I., the proposed study will be conducted by a team, which consists of the Functional Genomics Core Facility at MSM, and Dr. Ghaemi, Tufts Medical Center, MA. The proposed study will be an element within our bipolar disorder sleep-wake cycle study, in which we already have access to skin samples of bipolar patients.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A goal of our present proposal is to establish an assay system by which circadian rhythms can be monitored using human fibroblasts with a circadian reporter and real time bioluminescence monitoring, and to test our hypothesis that clinical response to lithium can be predicted using human peripheral cells. b. Studies and Results. The goal of the first Aim is to apply in vitro circadian rhythm recording method using human primary fibroblasts. So far eleven healthy participants were recruited and six fibroblast cell lines have been established. After cultivation and expansion, the circadian clock Bmal1 promoter-luciferase gene was delivered to fibroblasts, and Bmal1-luciferase cell lines were established. We optimized culture and recording conditions. Robust and sustained circadian rhythms have now been recorded in these human fibroblast cell lines. Average period length was 24.60 [unreadable] 0.16 h (N=5). The results were in agreement with those previously published by Dr. Brown and his colleagues (Brown et al., 2005) and, therefore, we have completed the Aim 1. In Aim 2 we plan to examine the effects of lithium on circadian rhythm parameters in fibroblasts obtained from lithium responders and non-responders. We have been optimizing lithium treatment conditions to be applied to bipolar disorder patients'cells. Since high lithium concentrations were used to test the effects of lithium on the period length in previous studies, we tested 3, 5, 7, and 10 mM lithium stimulation on the period length. At the same time, we want to test the effects of lithium at therapeutic concentrations, e.g., 1 mM. Since acute 1mM lithium stimulation did not show significant impact, we have been culturing cells in the presence of l mM lithium for one or four weeks before rhythm recording. We chose four weeks as well because the clinical lithium sensitivity is first determined four weeks after lithium therapy.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Bipolar disorder, a common psychiatric disorder, is characterized by recurrent episodes of mania and depression. The mood stabilizer lithium has proven its efficacy in preventing the recurrence of mania and suicide more than any other drug for more than 50 years. In the past decade, however, lithium use in the United States has declined substantially because lithium is more difficult to use and has a low margin of safety. Only a subpopulation of bipolar patients shows benefits from lithium treatment. To improve lithium pharmacotherapy, it is beneficial to develop an in vitro assay that can be applied prior to treating patients with the drug to predict which patients will show a therapeutic response to lithium. The assay can also be used to find drugs that are as effective as lithium, applicable to all types of bipolar patients, and with fewer side effects. Because there is no cell or animal model to study differential lithium sensitivity, such assay can be used as a model to understand mechanisms how such sensitivity is controlled. Recent studies indicate that multiple genes and proteins cooperate to control pathogenesis of bipolar disorder and lithium sensitivity. Therefore, testing for differential lithium responsiveness utilizing more comprehensive parameters should be more successful in predicting lithium sensitivity. Two parameters are chosen to compare lithium sensitivity;in vitro measurements of circadian phase and peroxiredoxin 2 (PRD2) in blood cells. There is a close relation between mood status and circadian rhythm expression, and lithium has been shown to affect circadian rhythm expresion in many species, including humans. Therefore, circadain parameter is one of the promising output to evaluate the effects of lithium on moode stabilization. Another line of study has shown that bipoalr patients have neurodegenration in their brains, and lithium can fix such conditiosn. Our previous R-Center supported project identified PRD2 as a single gene that was affected by lithium stimulation. Considering PRD2'neuroprotective roles, it would be worth evaluating the effects of lithium on this gene expression between lithium responders and non-responders. In this proposal, lithium responses in these parameters will be correlated to test the hypothesis that lithium differentially affects circadian phase and PRD2 levels in macrophages of lithium-responsive and lithium-insensitive patients.