Ying-Hui Fu - US grants

DESCRIPTION (Investigator's abstract): Autosomal dominant leukodystrophy (ADLD) is a rare adult-onset demyelinating disorder. We have identified 6 families with this disorder. Two of these are large pedigrees for whom a tremendous amount of clinical, neuroradiological and neuropathological data has been collected. Although these patients share many clinical features with other white matter disorders, unique neuropathological findings suggest that the genesis of this disorder neither resides in defects of structural myelin proteins nor fatty acid metabolism in peroxisomes. ADLD is not an immune disease like multiple sclerosis (MS). We've demonstrated that lesions in ADLD brain have dramatic reduction in astrocyte number and that the surviving astrocytic cells are morphologically very abnormal. We hypothesize that ADLD results from a defect that interferes with a unique element in the myelination process and that understanding of this defect may provide novel insights into the process of myelin maintenance and turnover. We have localized the gene causing ADLD in these two large families to chromosome 5q3 1. Fine mapping has further narrowed the region and a complete physical map predicts the gene to reside within 3 megabases, much of which has already been sequenced. Candidate gene identification and testing are underway. Some genes in the region have already been eliminated using various mutation analysis strategies. Several plausible candidates are currently being tested including a novel gene with multiple EGF-like domains. This proposal outlines a strategy for identifying and characterizing the gene. Available patient material, physical mapping reagents and genomic sequence position us well for accomplishing this goal. In addition, experiments will be pursued toward preliminary characterization of both the wild-type and mutant ADLD protein. Understanding the cause of this demyelinating disorder may yield clues to genetic factors that modulate the expression of acquired leukodystrophies. Ultimately, discovery of a new element in the synthesis and maintenance of myelin may provide a novel target for compounds that may stimulate remyelination in more common disorders like MS.

[unreadable] DESCRIPTION (provided by applicant): Familial Advanced Sleep Phase Syndrome (FASPS) is the only known Mendelian phenotype of the human circadian system. We've identified and characterized the clinical phenotype and identified five genes that, when mutated, cause FASPS. Two of these, casein kinase I? & ? (CKI?/?), are recognized to harbor mutations that segregate with FASPS in families and lead to decreased activity in vitro. A mutation in a third gene, period 2 affects a CKI?/? phosphorylation site. Work in a number of laboratories has characterized some substrates of these kinases, but a comprehensive and unbiased method for identifying substrates has been impossible given the large number of kinases and phosphatases present in any cell or organism. We will employ an innovative chemical genetic approach to specifically label substrates of these enzymes by engineering mutations into the ATP binding pocket. Reciprocal chemical modifications of ATP are engineered to synthesize ATP analogs that can only be accommodated by the mutated (analog-sensitive) kinases. This will provide a more complete compendium of substrates for CKI?/? and will allow assessment of the redundant and unique functions of each enzyme. This approach will also be applied in identifying multiple phosphorylation sites on known substrates by these kinases. In vitro biochemical assays can be performed to monitor specific effects of the FASPS mutations on each of these substrates. Next, transgenic mice will be generated to carry a BAC with each gene harboring the analog-sensitive mutations. These will be crossed onto null backgrounds and will represent mice with near normal kinase activity since the analog-sensitive kinases still accept, and transfer phosphate groups from ATP. Mice carrying analog sensitive mutations for both CKI? and CKI? will be generated. We can then rapidly and reversibly inactivate these kinases through use of chemical inhibitors that bind specifically in the analog-sensitive ATP binding site. These mice will be studied at different developmental time points to monitor the phenotype when one or both kinases are inactivated. In particular, we will focus on the circadian system but are also interested in whether the lethality that is seen in the CKI? knock out mice is the result of its effects on development or of its activity throughout the life of the mouse. This work will result in identification of many CKI?/? substrates and molecular dissection of the role of these kinases in human circadian rhythmicity. Identification of substrates and dissection of particular pathways in phenotypes such as circadian rhythmicity will have profound implications for therapeutics of circadian phenotypes and understanding of physiological mechanisms. [unreadable] PUBLIC HEALTH RELEVANCE: CKI? and CKI? are important kinases for many essential biological functions. This proposal outlines a plan to elucidate the normal role of CKI? and CKI? through identification of their substrates and studies aimed at understanding substrates that are important for the functional consequences of CKI?/? in circadian rhythm. We will also examine phenotypes resulting from reversibly inactivating these kinases in vivo. [unreadable] [unreadable] [unreadable]

Demyelinating and dysmyelinating disorders include a large number of degenerative conditions in humans including Multiple Sclerosis. Recently, myelin involvement in psychiatric disorders such as schizophrenia was also suggested. Understanding how myelin is synthesized and properly maintained is a challenging task that has been under intense investigation for decades. However, our knowledge of these processes is still limited and effective treatments for most of the demyelinating/dysmyelinating diseases are absent. Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive, neurological disorder characterized by symmetrical widespread myelin loss in the CNS, with a phenotype similar to chronic progressive multiple sclerosis (MS). We have recently identified a genomic duplication that causes ADLD. Patients carry an extra copy of the gene for the nuclear lamina protein, Lamin B1, resulting in increased gene dosage in ADLD brain tissue. Increased expression of Lamin B1 in Drosophila resulted in a degenerative phenotype. In addition, an abnormal nuclear morphology was apparent when cultured cells over-expressed this protein. This is the first human disease attributable to Lamin B1 mutations. Antibodies to Lamin B are found in autoimmune diseases and it is also an antigen recognized by a monoclonal antibody, J1-31, raised against plaques dissected from MS patient brains. This raises the possibility that it may be linked to the autoimmune attack that occurs in MS. In this grant, we propose to study the expression pattern and regulation of Lamin B1 to lay a foundation for future investigation into the mechanism by which Lamin B1 regulates proper myelin maintenance. We have generated BAC and cDNA transgenic mouse models that express less than four extra copies of LMNB1 genes. We are also in the process of generating conditional (under tet regulation) overexpression mouse models. Characterization of these mice will provide insight into the mechanisms not only for ADLD pathophysiology but also for myelin biogenesis. In addition, we identified miR-23 as a negative regulator of Lamin B1. We propose to use primary culture and co-culture methods to study the effect of Lamin B1 and miR-23 in oligodendrocyte development and myelination. Such knowledge will yield insights into pathways through which Lamin B1 overexpression leads to demyelination. Understanding the mechanism of this demyelinating disorder may provide clues to pathways that modulate the expression of acquired leukodystrophies. Ultimately, this knowledge will provide new insight in the synthesis and maintenance of myelin and identify novel targets for developing therapeutic interventions for stimulating remyelination in common disorders like MS.

DESCRIPTION (provided by applicant): Sleep is a global state and its control mechanisms are manifested at every level of biological organization- from genes and intracellular mechanisms, to networks of cell populations, to phenotypes at the organismal level. They include (but are not limited to) arousal, motor control, autonomic function, behavior, and cognition. We live in a sleep deprived society. Prolonged sleep loss impairs temperature control, dietary metabolism, immune function, and eventually leads to death. Sleep deprivation increases an individual's risk of cancer, metabolic syndrome, psychiatric, and other disorders. Understanding the biological basis of sleep in humans is an extremely difficult challenge since the biological determinants of our sleep are affected by behavior and other factors including life-style choices, socio-economic status, health, employment, school, and exogenous chemicals like caffeine and alcohol. Sleep and circadian function are distinct processes that interact in living organisms. Sleep is controlled by at least two processes: a circadian pacemaker (the clock) ticking with periodicity of ~24 hours, and a homeostatic drive that increases during wakefulness and dissipates during sleep. Despite of the fact that we spend around one third of our life in the state of sleep, we understand almost nothing about regulatory mechanisms governing sleep quantity. A unique opportunity presented itself when we identified independent families with a dramatically reduced biological need for sleep. Identification of new subjects and expanding our collection of these families will establish a foundation for us to begin probing the molecular regulatory mechanisms of sleep homeostasis. Recently, we reported the first mutation that causes this short-sleep phenotype. Interestingly, this mutation gave a similar short sleep phenotype in human, mouse, and fly. All the genes identified in this study will therefore become entry points for us to unravel the enigmatic sleep related mechanisms. Ultimately, combining the knowledge from studies in multiple genes and in humans and model organisms will lead to a better understanding of sleep and its relationship to health and disease.

Abstract Health problems caused by sleep disruption and/or insufficiency have been recognized and documented in a report from the Institute of Medicine in 2006. This has emerged, to a large extent, from the pervasive social- networking and rapid information exchange world that came upon us in recent decades. The increasing usage of electro-digital devices after dusk negatively affects sleep quality and quantity. In addition, the need for shift- work schedule to suit the 24/7 society and for the frequent transmeridian travels have both increased dramatically in the last few decades. Hence, sleep deprivation and disruption have become inevitable for many people in the societies that we live. Concurrently, our world is facing a greater challenge in maintaining our brain healthy. Poor sleep has been suggested to contribute to dementia such as Alzheimer's Disease. Recently, we have found a mutation from humans whose sleep need is less than the general population. This mutation, when engineered into mice, also enables these mice to sleep less than control mice. These results imply that mouse is an excellent model for studying relevant molecular mechanisms. We also have established many useful reagents and mouse models in the last several years and are now in a position where we can apply these resources and methodologies to investigate these mechanisms. Currently, the theories of the functions of sleep include energy regeneration and allocation, synaptic plasticity maintenance, and neurotoxin clearance, etc. One of the most intriguing questions that arise from observing the natural short sleepers who we have been studying for a long time is that they don't seem to have negative health consequence (cognitively and physically) from life-long sleeping less. We hypothesize that the natural short sleeper mutation somehow endows the brains of these human subjects the ability to perform whatever necessary functions occur during sleep in a much more efficient manner and can complete these daily tasks in a shorter period even though these people stay awake longer than regular people each day. Alternatively, the brains of these natural short sleepers may not accumulate as much waste products during wakefulness as the regular sleepers. These possibilities are not mutually exclusive. Here we propose to investigate these possibilities with our mouse models. We will investigate whether the mice carrying this human short sleep mutation can execute neurotoxin clearance more efficiently and/or accumulate less neurotoxin than control mice. We will determine the specific cell types that are participating in DEC2's (the short sleep gene) function in the brain. We will also investigate the inter-relationship between DEC2 and Hypocretin, which plays a critical role in sleep-wake maintenance and A? clearance. We have outlined three independent yet complementary aims in this grant. The results of these studies will pave the way for future investigations of mechanisms/networks that interact to regulate healthy brain functions during sleep. The outcome of these studies will provide novel opportunities for therapeutic intervention of sleep-related disorders and innovative aids for recovery functions of sleep.

Abstract Sleep is essential for the maintenance of our cognition and neurological functions, and both quality and quantity of sleep are critical. We likely have known this for the entire human history. Yet, we remain astonishingly ignorant on how the quality and quantity of sleep are regulated. Excitingly, nature has provided us a very small number of human subjects who are genetically wired to sleep shorter hours per day (thus more efficiently). These people usually live a long and healthy (both physically and mentally) life. Identification of genetic changes in these people provides us concrete and specific molecules that are in the sleep duration/efficiency pathway. These molecules offer opportunities to not only map brain regions and cells for sleep regulation but also will lead us to gain understanding of neurocircuitry of sleep duration/efficiency. In this proposal, we will use integrated approaches to understanding how neurocircuit activities work in concert to regulate sleep duration/efficiency. Our hypothesis is that there exist unique neurocircuits for sleep duration and efficiency that are separate from the circuits for sleep-promoting and wake-promoting. Our experimental design outlined here is based on this hypothesis to reveal these circuits in a systematic way. We will first identify specific cell types with our gene-specific Cre mice. We will then generate a functional circuit diagram by mapping their projections. The role and function of these cells in sleep regulation will be tested in the context of circuit by linking the activity of these cells to sleep with precise interventional tools that change neural circuit dynamics. The results from this study will reveal how dynamic patterns of neural activity are transformed into efficient sleep. We will simultaneously monitor sleep state with EEG/EMG recording while actively recording and manipulating dynamic patterns of neural activity of specific cells. The results obtained from this study will provide a fundamental understanding of brain circuits for sleep duration/efficiency maintenance. Since quality sleep is the basis of healthy brain (cognitive and neurological function), understanding of how quality sleep circuit is obtained will not only shed new light on how poor sleep can lead to unhealthy brain but also give insight into mechanisms for treating brain dysfunctions.