Mechanisms of Circadian Rhythmicity in CLOCK-Deficient Mice

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DESCRIPTION (provided by applicant): The proposed studies will use a mouse model to investigate causes for two conditions, infertility and arthropathy. Infertility affects ~ 10% of couples, with about half of this related to male infertility. Abnormal formation of bone-like substances in tissues that are normally not calcified (arthropathy) interferes with tissue function limits motion, and is often painful. Forms of ectopic calcification range from heterotopic ossification (occurs with aging, following trauma, and in genetic and idiopathic forms), to age-related aortic stenosis, and aortic valve calcification (which is a prominent complication limiting surgical repair of the heart and cardiac valves). Understanding the process of ectopic calcification thus has significant potential therapeutic benefit. Mice with disruption of two genes involved in circadian rhythm generation, CLOCK and NPAS2, not only lose their circadian rhythms, but they are also infertile and developed a severe, age-related and progressive ectopic calcification. CLOCK and NPAS2 normally partner with BMAL1 in regulating circadian rhythms; BMAL1- deficient mice are also have reduced fertility and progressive arthropathy. These pathological phenotypes do not occur in animals lacking circadian rhythms due to other genetic lesions, indicating that it is disruption of the CLOCK/NPAS2:BMAL1 transcriptional complexes, likely unrelated to circadian clock function, that allows these pathologies to develop. Studies of animals with gene disruption throughout the body are complicated by the multiple potential sites of action of these genes, ranging from potential effects on reproductive endocrinology and behavior to defects in spermatogenesis (for infertility), and multiple potential cell types of the musculo-skeletal system (for arthropathy). We will thus use advanced genetics approaches to produce tissue-specific genetic lesions, to identify and isolate the cell types involved in fertiliy regulation and in arthropathy prevention, allowing assessment of the molecular events that lead to the pathological conditions. Our overall hypothesis is that local dysregulation of gene expression in animals lacking CLOCK and NPAS2 (or BMAL1) allows these pathological conditions (calcification in specialized but poorly defined cell types at the junction of cartilage and tendon to bone, and infertility). Our overall objective is to define the cell types involved using an unbiased, genetic approach in vivo, and then use these same approaches to label the cell types involved and to isolate them for molecular analysis at times when the genes are critically important for preventing these phenotypes. Identification of the cellular and molecular events occurring in this very reproducible, genetic model in mice should contribute to understanding and ultimately regulating fertility, and for understanding and ultimately preventing pathological ectopic calcification in humans.