David K. Welsh, MD/PhD - US grants

DESCRIPTION (provided by applicant): Daily oscillations in mammalian physiology and behavior persist even in a constant environment, and their disruption leads to jet lag, sleep disorders, and other maladies. Such "circadian" (ca. 24-hr) rhythms are driven by a biological clock located within the brain, in the suprachiasmatic nucleus (SCN). SCN cells express "clock genes," components of a molecular feedback loop comprising the clock. Most of these, including Per1, oscillate in the SCN. Clock gene expression also oscillates in some peripheral tissues, but damps out after a few days without the SCN. The candidate recently completed M.D./Ph.D. training at Harvard and residency training in psychiatry at the University of Pittsburgh. His Ph.D. thesis work provided key evidence for single cell clocks in the SCN that generate independent circadian oscillations in a culture dish. The objective of this proposal is to enable the further career development of the candidate into an independent scientist with expertise in modern molecular and cellular neurobiology. This will be accomplished through a mentored research project which is a natural extension of the candidate's thesis work, but employs a different range of methodology, including transgenic mice, genetically encoded fluorescent reporters, live cell imaging, and DNA microarrays. Cells dissociated from a Per1-GFP transgenic reporter mouse will be used to determine (1) whether single SCN neurons are truly independent clocks, (2) whether damping of rhythms in peripheral tissues is due to single cell damping or desynchrony, (3) the mechanisms of functional coupling among SCN clock cells, and (4) how functional subtypes of SCN clock cells differ by morphology, immunolabeling, and gene expression profiling. The candidate will have a position in the Department of Psychiatry at UCSD, and initially will work in the research mentor's lab at Scripps Research Institute. This environment will provide an outstanding neuroscience community, a mentor with vast experience and resources related to the methodology proposed, and opportunity for future integration with clinical work in psychiatry.

Pneumocystis pneumonia; drug resistance; diagnosis design /evaluation; communicable disease diagnosis; diagnostic tests; sulfamethoxazole; trimethoprim; genetic strain; longitudinal human study; genotype; disease /disorder prevention /control; clinical research; human subject;

[unreadable] DESCRIPTION (provided by applicant): Daily oscillations in mammalian physiology and behavior persist even in a constant environment, and their disruption leads to jet lag, sleep disorders, and other maladies, including mood disorders. Such "circadian" (ca. 24 hr) rhythms depend on a biological clock located within the brain, in the suprachiasmatic nucleus (SCN). Most cells express "clock genes", components of a transcriptional feedback loop comprising the intracellular clock, but the SCN is the master pacemaker: it has access to synchronizing light/dark input from the retina, specialized coupling mechanisms to maintain coherence among its component cellular oscillators and enhance its robustness, and neuronal efferent projections to synchronize cellular oscillators in peripheral tissues throughout the body. Recent work, however, has challenged the simplistic view that SCN neurons are all stable, autonomous, single cell transcriptional feedback oscillators. The objective of this proposal is to define the autonomy, persistence, and precision of SCN and fibroblast circadian clock cells, and to explore the interdependence of intracellular transcriptional, electrical, and calcium rhythms in these cells. This will be accomplished using mechanical, pharmacologic, and genetic approaches to disrupt cell interactions and manipulate membrane potential or intracellular calcium. Effects on the intracellular circadian clock will be assessed in individual cells by using optical methods to measure calcium and clock gene transcription, and multielectrode arrays to monitor neuronal firing. Specifically, we will test the hypotheses that: (1) SCN neurons require tonic (but not rhythmic) input from other neurons to maintain rhythmicity, (2) apparent non-rhythmicity of some SCN neurons is a stochastic event due to membrane hyperpolarization rather than a reflection of a stable non-rhythmic subtype, and (3) cells require a tonic level of calcium (but not rhythmic calcium) for transcriptional or electrical rhythms. Answers to these fundamental questions about the cellular basis of circadian rhythmicity will be essential for an understanding of how circadian clocks contribute to health and disease, and serve as a basis for novel therapeutic approaches. PUBLIC HEALTH RELEVANCE A biological clock in the human brain keeps track of time of day and orchestrates countless circadian (ca. 24 hr) rhythms throughout the body. By further delineating the mechanisms of this clock at the level of single cells, the experiments proposed here may suggest new therapeutic approaches not only to jet lag, shift work, and other sleep disorders, but also to cancer, diabetes, and depression. [unreadable] [unreadable] [unreadable]

Summary Alzheimer?s disease (AD) is a devastating neurodegenerative disorder affecting the lives of more than 5 million Americans and their families, and is the biggest forthcoming health challenge. AD is a multifactorial disorder manifested clinically by progressive memory loss, decline in cognitive functions and ultimately leading to dementia. Despite being the subject of intense research, there is no cure for AD, therefore, identifying therapies that can reduce disease progression at early stages is critical. Circadian impairment is a major feature of Alzheimer?s disease. Behavioral circadian alterations, known as sundowning, are experienced by more than 80% of patients and represent the leading factor for hospitalization and nursing home placement in AD. New research suggest that circadian disruption occurs early during disease progression and contributes to neurodegeneration, but the pathways that mediate the effects of clock dysfunction on AD pathophysiology are not well characterized yet. The proposed work will investigate the epigenome as the target of circadian deregulation. Circadian rhythms are generated by oscillation of clock genes, including BMAL1, in transcription/translation feedback loops and epigenetic mechanisms are deeply involved in their regulation. We previously reported alterations in rhythmic DNA methylation of clock genes in AD brains and identified BMAL1 as a regulator of methylation. While ample evidence demonstrates the disruption of DNA methylation in AD, the potential cross- talk between circadian dysfunction and epigenetic alterations in mediating AD pathology is yet unknown. We now propose to apply a step-wise approach starting by the identification of alterations in chromatin dynamics dictated by the circadian clock; and narrowing down to the expression of specific genes whose deregulation may trigger neurodegeneration. This work will apply cutting-edge technology to generate temporal maps of genome-wide chromatin accessibility, DNA methylation and transcription in the mouse brain in the context of circadian disruption and AD pathology. In addition, we will evaluate the mechanisms that mediate the beneficial effects of the small molecule Nobiletin (NOB) in AD mouse models, focusing on NOB activity as circadian-enhancer. These studies will define NOB targets of action in the brain and will re-evaluate their potential as disease modifying therapy. Understanding these molecular pathways may unravel novel targets and timing for improved interventions. As treatments curing or even postponing AD remain yet elusive, prolonging patient independence and daily functioning might represent a major option in clinical care.