Robert H. Miller, PhD - US grants

This research proposal is an experimenal investigation into (1) the identification of synaptic transmitters, (2) the physiological basis of transmitter action at the single cell level, and (3) the morphological identification of physiologically and pharmacologically characterized cells. Inhibitory and excitatory transmitter agonists and antagonists are added to the bathing medium using perfused-retina eyecup preparations of rabbits and several types of amphibians (mudpuppy and tiger salamander). Analysis of drug action is based on intracellular electrode experiments using conventional recording techniques, single electrode voltage clamp techniques ad a lock-in amplifier for quantitative measurements of conductance changes. A similar, parallel set of experiments will be carried out using enzyme dissociated cells maintained in tissue culture conditions. Intracellular recording of single dissociated cells will be analyzed as agonists/antagonists are added to the bathing medium, iontophoretically applied, or introduced through pressure injection. The isolated retina of amphibians and rabbits will be studied for types and levels of amino acids released into the bathing medium, using reverse-phase high performance liquid chromatography. Particular emphasis will be placed on the identity of rod and cone transmitters. These experiments will also be combined with tissue culture techniques to fractionate the retina into limited neuronal populations, through the use of neuroactive ligands. The goal of this research is to identify synaptic transmitters and associate them with specific pathways and cell types. Several disease states which afflict the retina (retinitis pigmentosa; macular degeneration), show prolonged states in which photoreceptor function is compromised while leaving a more or less intact retina. An understanding of the chemicals involved in photoreceptor transmission, together with the synaptic receptors which mediate their action may help in the design of drug therapy to assist patients with limited vision.

The proposed investigations are designed to identify environmental factors that support axon growth during experimentally induced axon tract formation in the developing mammalian CNS, and to determine if those, or similar factors can be implicated in the failure of regeneration in adult animals. In earlier studies we have shown that nitrocellulose implants coated principally with astrocytes from young animals are able to support axon growth in neonates, and when transplanted, suppress scar formation and promise directed axon growth in the mature mouse forebrain. Implants coated with astrocytes from mature animals fail to support axon growth in either developing or mature animals. To identify the cellular and molecular properties of purified astrocytes important in the support of axonal growth, we will first purify astrocytes from implants of different ages, and compare the capacity of young against old astrocytes to support axonal elongation "in vitro". Second, we will assess whether this change from young to old is an intrinsic property of the astrocytes or depends on environmental factors. Third, we will determine if astrocytes preferentially support axonal growth from neurons of the same, rather than remote regions of the brain. Fourth, monoclonal antibodies will be generated that distinguish between young and old astrocytes; and used to influence axonal growth on astrocytes "in vitro", will be used to investigate the molecular basis of the axon/astrocyte interaction through biochemical characterization of their antigens. These studies will provide a better understanding of the role of astroglia in the regulation of CNS axonal growth.

During development, axons grow long distances in the central nervous system to contact their specific targets, while in the adult, there is little axon growth following a lesion and functional recovery is limited. Immature astrocytes have been proposed to be the cellular substrate for axon growth during development, and after a lesion to the adult CNS, "reactive" mature astrocytes are a major cellular component of glial scars that have been proposed to block axon growth. These observations suggest that the capacity of astrocytes to support and direct growth alters during CNS maturation. The molecular basis underlying the ability of astrocytes to support axon growth during development, and its modulation during astrocyte maturation is unknown. This study is designed to determine if changes in the interactions both between astrocytes and neurons; as well as between astrocytes and astrocytes, are responsible for the inhibition of neurite outgrowth by astrocytes after maturation. Dr. Miller will identify molecules involved in the following changes observed when astrocytes mature: (1) Neurite outgrowth is reduced on mature astrocytes, (2) Neurons have decreased adhesion to mature astrocytes, and (3) Astrocyte/astrocyte adhesion increases in mature astrocytes. In addition, neurite growth will be compared in a 3-dimensional matrix of immature or mature astrocytes, and the perturbation of cell interactions involving astrocyte/neuron and astrocyte/astrocyte adhesion in this system will be determined. These studies will provide new information on the regulation of axon outgrowth by astrocytes of the developing and adult CNS.

Astrocytes of the vertebrate CNS have been proposed to perform a wide variety of different functions including acting as a substrate for axon growth during development, contributing to the induction of the blood brain barrier and forming glial scars following injury to the adult CNS. Our recent studies suggest that astrocytes are a heterogeneous class of cells. In the proposed studies we will examine astrocyte diversity in the rat spinal cord and test the hypothesis that distinct types of astrocytes perform distinct functions in the developing, adult and injured spinal cord. Three different approaches will be used to define astrocyte diversity in the developing spinal cord: single cell cloning, retrovirus mediated gene transfer and the generation of cell type specific monoclonal antibodies. Initial studies suggest that cultures of neonatal rat spinal cord contains five morphologically distinct types of astrocytes. We will determine the time of origin, and the factors, that regulate proliferation and differentiation of each of these different cell types. Retrovirus mediated gene transfer will be used to determine the cell lineage relationships of the different types of astrocytes, both in cultures containing all classes of spinal cord cells and in the intact developing spinal cord. Recently isolated, as well as novel cell-type specific monoclonal antibodies will be used to distinguish biochemical differences between these cell types and to localize them in the intact spinal cord. The functional potential of the different types of astrocytes will be examined by analyzing their ability to support neurite outgrowth from developing neurons, suppress glial scar formation and reestablish the blood brain barrier after injury in the adult. The extent of neurite outgrowth and its molecular basis on the different populations of astrocytes will be established using purified cell populations in vitro. Transplantation of nitrocellulose implants coated with the different populations of astrocytes in to the adult CNS will be used to assess the ability of each type of cell to suppress glial scar formation and re-establish the blood brain barrier after injury. These studies will provide new, and important information on the extent and functional significance of astrocyte diversity in the spinal cord. Since the response of astrocytes to CNS injury is important for functional recovery it is critical to define specific astrocytic cell types in the CNS and to describe their individual functions following a CNS lesion. Such information is essential if we are to understand and subsequently manipulate the complex astrocytic response to CNS injury.

This research proposal is an experimenal investigation into (1) the identification of synaptic transmitters, (2) the physiological basis of transmitter action at the single cell level, and (3) the morphological identification of physiologically and pharmacologically characterized cells. Inhibitory and excitatory transmitter agonists and antagonists are added to the bathing medium using perfused-retina eyecup preparations of rabbits and several types of amphibians (mudpuppy and tiger salamander). Analysis of drug action is based on intracellular electrode experiments using conventional recording techniques, single electrode voltage clamp techniques ad a lock-in amplifier for quantitative measurements of conductance changes. A similar, parallel set of experiments will be carried out using enzyme dissociated cells maintained in tissue culture conditions. Intracellular recording of single dissociated cells will be analyzed as agonists/antagonists are added to the bathing medium, iontophoretically applied, or introduced through pressure injection. The isolated retina of amphibians and rabbits will be studied for types and levels of amino acids released into the bathing medium, using reverse-phase high performance liquid chromatography. Particular emphasis will be placed on the identity of rod and cone transmitters. These experiments will also be combined with tissue culture techniques to fractionate the retina into limited neuronal populations, through the use of neuroactive ligands. The goal of this research is to identify synaptic transmitters and associate them with specific pathways and cell types. Several disease states which afflict the retina (retinitis pigmentosa; macular degeneration), show prolonged states in which photoreceptor function is compromised while leaving a more or less intact retina. An understanding of the chemicals involved in photoreceptor transmission, together with the synaptic receptors which mediate their action may help in the design of drug therapy to assist patients with limited vision.

DESCRIPTION (Adapted from the applicant's abstract): The aim of this proposal is the identification and characterization of the mouse severe combined immunodeficiency (scid) gene. Mice carrying the scid mutation, SCID mice, are deficient in double strand DNA repair, representing one of the rare viable mammalian models of a DNA repair system. SCID mice are profoundly deficient in mature lymphocytes due to failure of DNA recombination of the antigen receptor genes. The mice have proven to be invaluable for studies of lymphoid ontogeny and establishment of models of infectious diseases for vaccine development. Detailed analysis of the function of the scid gene in disease and in health is clearly warranted, but has been hindered by the lack of success trying to isolate the gene. This proposal outlines an approach to isolate the scid gene by positional cloning. This approach has not been directly applied to the identification of the scid gene, but has been used successfully to isolate several important human disease genes. The applicant has established a high resolution linkage map of the genomic region containing the scid locus based on the analysis of nearly 300 backcross progeny. The applicant has identified three molecular markers that do not recombine with the scid phenotype and, therefore, must be physically close to the scid gene. The applicant has used these markers to screen several mouse and human Yeast Artificial Chromosome (YAC) libraries and has identified multiple DNA clones. The first goal of this proposal will be to identify a YAC clone that contains the scid gene. This will be achieved using alternative approaches. The applicant has a system by which YACs can be assayed directly for their ability to complement the scid defect in vitro. If none of the YACs contain scid by this assay, additional overlapping YACs will be isolated to increase the genomic coverage. These YACs will be used to generate a physical map of the region of the genome containing the scid locus. The physical map of the region will be extended until recombination points, which flank the scid locus, are crossed. Once this is achieved, the applicant will be confident that the scid locus is contained within the contiguous set of YAC clones. YACs that must contain the scid locus will be used to identify expressed genes by hybridization to cDNA libraries and Northern blots, and analysis for CpG islands. Homologous human YACs will be used to identify regions of conserved sequence between human and mouse, which are likely to be candidate genes. Once the scid gene is isolated, it will be characterized for sequence homology to other proteins and the expression in normal development and in response to DNA damage. In addition, the homologues of scid will be isolated from other species for comparative and evolutionary analysis.

DESCRIPTION (provided by applicant): Understanding the cellular and molecular mechanisms that regulate the generation of oligodendrocytes, the myelinating cells of the vertebrate central nervous system is essential both to establish a comprehensive vision of neural development and to effectively generate new therapeutic approaches towards demyelinating diseases. In the last funding period we have made substantial gains in defining the location and molecular signaling that regulates the appearance of oligodendrocyte precursors and their dispersal in the developing spinal cord. In the current proposal we outline a series of approaches that are designed to further our understanding of the cellular and molecular interactions that promote later stages of oligodendrocytes development and to relate that information to the regulation of successful myelin repair in the adult CNS. Building on new approaches and recent preliminary data we outline 3 specific aims that investigate distinct but related aspects of oligodendrocyte development. In the first aim we will test the hypothesis that mature MBP+ oligodendrocytes influence the timing and success of spinal cord myelination as the direct producer of myelin and through feedback signals to OPCs. In addition we will determine whether a local postnatal depletion of MBP+ oligodendrocytes compromises myelin repair in the same region of the adult following a second insult. In the second aim we will characterize the role of GFAP+ astrocytes in the generation of the spinal cord oligodendrocyte lineage and myelination and the third aim will address the role of both mature oligodendrocytes and astrocytes in the control of remyelination in the adult spinal cord. These studies take advantage of a novel approach developed in the laboratory during the last funding period in which we are able to selectively eliminate distinct populations of mature oligodendrocytes and astrocytes in precise locations at any point in the developing and adult CNS. To accomplish this we have generated a series of transgenic animals in which we express an inducible caspase 9 (iCP9) off cell type specific promoters. Induction of the iCP9 through cross-linking with a small molecule variant of the cell and tissue permeable FK506 stimulates apoptosis specifically in the targeted population of cells. These animal models provide us with a unique opportunity to assess the consequences of selective cell loss on myelination and myelin repair in the intact vertebrate CNS. The data generated during the course of these studies will provide new directions for the development of therapies for demyelinating diseases such as multiple sclerosis.

DESCRIPTION: The purpose of this project is to characterize the role of GRO-a, an a-chemokine recently demonstrated by Dr. Miller's laboratory to be a major environmental factor that modulates the response of oligodendrocyte precursors to PDGF. In the first Specific Aim, the chemokine structural motifs required to enhance the PDGF proliferation response of oligodendrocyte precursors will be defined. In the second Specific Aim, the applicant intends to establish optimal conditions for interactions between GRO-alpha and PDGF to elicit oligodendrocyte precursor chemotaxis, and the capacity of GRO-alpha to enhance PDGF receptor signaling. In the third Specific Aim, molecular mechanisms of GRO-alpha action will be elucidated by evaluating ligand binding characteristics, as well as by determining the involvement of calcium and G proteins in signal transduction cascade. In the fourth Specific Aim, the distribution of biologically effective chemokines in the developing spinal cord will be determined.

Exercise therapy in healthy sedentary adults improves limb strength by about 30%, increases aerobic capacity (VO2max) by about 15%, reduces depression and anxiety, and increases mood and self esteem. An increasing body of evidence indicates comparable benefit occurs in disease states that are relevant to ALS, such as post-polio syndrome - a disease of the lower motor neuron for which increased strength and aerobic capacity has been demonstrated - and multiple sclerosis - a disease of the upper motor neuron for which improved strength, aerobic capacity, mood, and quality of life has been shown. Neither the efficacy nor the safety of exercise therapy for ALS, or any of the other neurogenic diseases represented by the MDA, is known. The specific aims of this study are to determine if a supervised 4-month exercise training program can safely improve muscle strength, aerobic capacity, pulmonary function, mental health, and qualify of life in patients with ALS. The design is parallel, randomized, controlled, and blinded, and the sample size is 40 patients. If exercise increases muscle strength by the same mean magnitude in ALS as for healthy sedentary adults, it would represent a gain comparable to the expected mean decline in muscle strength over 9 months of the disease course. The possibility also exists that in ALS psychosocial benefits may be found from exercise which will surpass the physiological ones (strength and aerobic capacity). The recent data from multiple sclerosis (MS) is encouraging wherein exercise therapy had a significant impact in improving quality of life, strength, and endurance. Exercise therapy is an inexpensive, universally available, therapeutic modality. Improvement by exercise therapy of any of these measures without adverse effect would be expected to have a significant impact on clinical practice, not only for ALS, but also for other neurogenic diseases causing weakness.

DESCRIPTION (provided by applicant): Myelination is a crucial process in vertebrate CNS maturation. Lack of myelination during development or loss of myelin in the adult leads to severe functional deficits. CNS myelin is produced by oligodendrocytes that develop in appropriate numbers and at appropriate locations to myelinate all long projection axons. The local control of oligodendrogenesis during development or following demyelinating diseases is not well understood. We recently identified a novel regulatory mechanism that appears to play a key role in regulating the development of oligodendrocytes and in myelination. The chemokine GRO-alpha, signaling through its receptor, CXCR2, regulates the proliferation of oligodendrocyte precursors in response to platelet derived growth factor (PDGF) and provides a potent stop-signal for oligodendrocyte precursor migration. In this renewal application we will use transgenic animals that either lack CXCR2 receptors, or overexpress the ligand GRO-alpha to define the function of GRO-alpha/CXCR2 signaling during oligodendrocyte development and in response to demyelination. To unambiguously identify oligodendrocyte lineage cells we will utilize transgenic animals in which these cells are specifically labeled through expression of Enhanced Green Fluorescent Protein (EGFP) driven off the PLP promoter. We will 1) test if the GRO-alpha/CCR2 pathway regulates development of oligodendrocytes and myelination in the spinal cord, by comparing the timing and degree of myelination between normal and CXCR2 null animals; 2) identify if GRO-aplha/CXCR2 signaling inhibits the migration and dispersal of oligodendrocyte precursors by transplanting EGFP+ cells into different environments as well as direct time lapse migration analyses of EGFP-labeled cells in slice preparations; 3) determine whether GRO-alpha/CXCR2 signaling enhances or inhibits remyelination in the adult CNS by comparing recovery from lysolecithin induced demyelination or induction of EAE in wild type and CXCR2-null animals. These studies will define GRO-alpha as an important regulatory molecule in oligodendrogenesis and a potential target for therapy in diseases of myelin, such as Multiple Sclerosis, and disorders of unregulated gliogenesis such as oligodendroglioma

DESCRIPTION (provided by applicant) Fetal ischemic insult to the developing CNS results in irreversible and devastating functional consequences such as those observed in cerebral palsy. A hallmark of such early ischemic damage is a dramatic reduction in myelinated white matter. We propose to use a powerful model of fetal ischemia combined with biochemical and immunological tools that identify distinct stages in oligodendrocyte maturation to define the precise events affected by fetal ischemic insults. Normal oligodendrocyte development involves a number of crucial regulatory events, only some of which appear compromised by fetal ischemia. We hypothesize, based on preliminary data, that the loss of white matter after ischemic injury reflects changes in regulatory molecules that control the proliferation, migration and survival of oligodendrocyte precursors. In the first aim we will identify the specific stages at which oligodendrocyte lineage cells are lost following fetal ischemic insult and test the hypothesis that the insult alters the expression of growth factors required for oligodendrocyte development. In the second aim we will test the hypothesis that prenatal ischemic insult reduces the proliferation and migration of oligodendrocyte precursors. In the third aim we will quantify oligodendrocyte precursor cell death following ischemic insult and test the role of several different identified regulatory molecules in the induction of oligodendrocyte cell death. These studies utilize a model of fetal ischemia to identify the injury-susceptible events in oligodendrocyte maturation that eventually result in loss of white matter. Completion of this project will identify candidate molecules that will be useful for the development of novel therapeutic interventions for treating a common and devastating pediatric CNS problem.

[unreadable] DESCRIPTION (provided by applicant): In this application we are requesting support for a selected subset of invited speakers as well as students and postdoctoral fellows to attend the 15th biennial meeting of the International Society for Developmental Neuroscience to be held in Edinburgh 4-7th of August 2004. Recent years have seen substantial advances in our understanding of the cellular and molecular mechanisms that regulate processes fundamental to development of the nervous system. This meeting will highlight specific areas of development where our advances in the basic understanding have begun to elucidate underlying mechanisms of disease and/or illuminate novel avenues for therapeutic approaches. For example, planned sessions range from animal models of Autism, through mitochondria alterations in the developing CNS to the role of stem cells in development and repair. The program is aimed at attracting basic scientists working in neural development as well as physician scientists working in areas of neurological disease. In addition, the long-term health of the field of developmental neuroscience is dependent on fostering young investigators and 2 sessions will be dedicated to young investigators with short talks selected from submitted abstracts. Furthermore, women will chair approximately one third of the general sessions. This is an exciting time in developmental neuroscience and the types of scientific interactions afforded by this meeting will help to advance our understanding by bringing together workers in distinct but related areas. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In this application we are requesting support for the seventh Gordon conference on Myelin to be held in II Ciocci, Barga, Italy from May 23-28, 2004. The field of myelin biology has seen significant advances since the last meeting including the characterization of transcription factors regulating critical steps in the development of myelinating cells, the identification of cellular mechanisms that control the behavior of remyelinating cells in diseases such as Multiple Sclerosis and the unraveling of the molecular complexes at axonal-glial junctions. The development of new animal models of demyelination promises to provide further critical insights into the normal function of myelin and its dysfunction in a variety of pathologies. The program for this 7th meeting has been developed to highlight these new advances in our understanding of the biology of myelin and myelinating cells and their role in disease. Care has been taken to minimize overlap in speakers with the previous meeting, or with related meetings in the same year and to expand the focus of the meeting beyond simply "myelin". The study of myelin and the biology of myelinating cells is; however, undergoing a period of expansion with the recruitment of scientists studying diverse aspects of membrane trafficking, cell signaling and cell migration. The program has been designed to attract scientists working both directly on myelin and its associated cells as well as in related areas of brain tumor biology and regeneration. This enhanced diversity will continue to increase the variety of myelin-associated scientist and provide for a dynamic and broad-spectrum meeting. To specifically encourage the participation of young investigators, registration and travel stipends will be offered as funds permit. All attendees will be expected to contribute to an oral presentation or present a poster. In addition to formal sessions, extensive poster sessions will encourage productive interactions between scientists with different expertise and in keeping with the goals of the Gordon Conference organization; such synergy will provide critical impetus and guidance to the field of myelin biology. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): In this application we are requesting support for the eighth Gordon Conference on Myelin to be held in Ventura California, USA between February 12th and 17th 2006. Our understanding of the biology of myelinating cells in the nervous system continues to expand rapidly. Since the last meeting in Italy in 2004 there has been a significant move towards utilizing the insights gained from the basic biology of myelinating cells in the development and understanding of the potential for myelin repair in the adult CNS. The realization that the adult CNS contains populations of stem cells and neural precursors capable of generating new myelin has propelled studies on the genesis and differentiation of myelinating cells to the forefront of biomedical research. The program for the 8th Gordon Conference on Myelin "Development and Diseases of Myelin" is designed to capitalize on these advances and further accelerate research in this area. We have attempted to reduce overlap with speakers of previous meetings and expand the areas that will be covered. The health of the field of myelin biology research depends on a dynamic group of investigators and this is driven largely by promoting young, newly independent scientists. We have made a concerted effort to invite investigators at the junior faculty level to be speakers at this meeting. The more senior investigators have been encouraged to act as session chairs and provide overviews of the field. This will allow the young investigators a unique opportunity to present their work, and become known to their scientific peers. Few other venues offer such an opportunity. We are also committed to fostering trainees in the field of myelin biology and to specifically encourage their participation we will offer stipends for travel and registration as funds permit. All attendees will be expected to contribute to an oral presentation or present a poster. We believe that poster sessions are a key element in fostering productive interactions between scientists with different expertise. To ensure extensive interaction at the poster sessions and between researchers at different levels of their career and with different expertise we will designate a group of senior scientist to act as "poster judges" and award a series of small prizes to those posters considered to be exceptional. In keeping with the goals of the Gordon Conference such interactions will provide impetus and direction to the field of myelin biology. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The prevailing notion that mammalian spinal cord oligodendrocyte precursors arise from restricted regions of the ventral ventricular zone as a result of direct signaling by sonic hedgehog appears to require revision. Dorsal regions of the spinal cord generate oligodendrocytes, as do multiple domains in more rostral CNS regions. Perhaps more compelling, oligodendrocytes can be generated in the total absence of Shh. We propose that commitment of cells to the oligodendrocyte lineage is regulated not by global signals but by local cell-cell interactions that preferentially occur in particular regions of the ventricular zone. Consistent with this hypothesis we have found that the appearance of OPCs is discontinuous along the rostral-caudal axis in the ventral ventricular zone and that interspersed between these cells are neuronal precursors suggesting that neuronally derived signals regulate the initiation of oligodendrogenesis. Support for this hypothesis comes from our recent studies demonstrating that retinal neurons are necessary and sufficient to promote oligodendrogenesis. In this proposal we will extend these studies to develop a clearer understanding of the environmental regulation of early oligodendrogenesis. In the first aim we will examine whether the ability to induce OPCs is a characteristic shared by multiple populations of neurons including those in the PNS, and characterize the signaling pathways responsible. Our preliminary data suggest that electrical activity is critical for the ability of neurons to induce oligodendrocyte precursors and in the second aim we propose to test this hypothesis in vitro and in vivo. How the population size of oligodendrocytes and their precursors are regulated during development is unclear. We propose that cell numbers are dynamically controlled to match environmental needs. To directly test this hypothesis in vivo we will use a novel cell type specific depletion approach to locally eliminate mature oligodendrocytes in aim 3 and OPCs in aim 4. Analyses of the responses to oligodendrocyte lineage cells to stage specific deletion will identify the major regulatory steps in oligodendrocyte development and provide novel targets for therapeutic intervention in demyelinating diseases such as Multiple Sclerosis. PUBLIC HEALTH RELEVANCE: Myelination is the fatty insulation that surrounds the processes of neurons and allows them to communicate rapidly with each other. In the brain and spinal cord, myelin is made by oligodendrocytes and they are essential to allow the adult nervous system to function correctly. How oligodendrocytes are formed is not well understood and the studies in this application will identify the environmental signals that promote their initial appearance and control the number of oligodendrocyte lineage cells in the mature central nervous system. Such information will provide new targets for the development of treatments for demyelinating diseases such as Multiple Sclerosis.

DESCRIPTION (provided by applicant): Schizophrenia is a complex, debilitating mental health disorder associated with significant morbidity and mortality. The molecular- and cellular-based mechanisms that contribute to schizophrenia remain undefined. Although schizophrenia has classically been considered a neurotransmitter-based disorder, there is emerging evidence that dysregulation of oligodendrocyte function is a key contributor to the mental deficits seen in afflicted patients. Currently there is not a tractable system that allows for the direct interrogation of the functional properties of neural cells types from patients with schizophrenia. Patient-specific sources of cells that are capable of robust and reproducible differentiation into specific neural lineages do not exist. We propose to develop a cell-based system whereby neural cells from afflicted individuals can be functionally assayed to interrogate the molecular mechanisms underlying schizophrenia. To achieve this goal we have developed a cutting-edge proposal that that incorporates the skill and expertise of multiple disciplines. In Aim 1 we will derive and characterize patient-specific, induced pluripotent stem (iPS) cells from juvenile-onset schizophrenia patients and controls. Since iPS cells are pluripotent, having the ability to differentiate into all cell types of the human body, in Aim 2 we will differentiate patient-specific iPS cells line into oligodendrocyte progenitor cells (OPCs) to provide a cellular source for oligodendrocytes. In the second phase of this project will characterize, compare, and functionally assay these patient-specific, iPS cell-derived oligodendrocytes from control and juvenile-onset schizophrenia patients using both in vitro and in vivo assays (Aims 3 and 4 respectively). We will also actively procure additional samples to derive and characterize patient-specific, iPS cells from juvenile-onset schizophrenia patients and controls during this second phase (Aim 5). There is great potential for patient-specific iPS cell technology to profoundly impact our understanding of human development and disease by providing genetically distinct, functional sources of human cells. By completing the aims set forth in this proposal we expect to provide a detailed characterization of oligodendrocyte function in patients afflicted with schizophrenia and provide insight into the pathophysiology of this complex disease. We have established an interdisciplinary team that combines strengths in clinical schizophrenia research, neural differentiation and function, as well as pluripotency and iPS cells to interrogate novel questions about the cellular and molecular dysfunction that contributes to schizophrenia. We expect that results from our studies will have immediate relevance to the understanding and treatment of this human disease. PUBLIC HEALTH RELEVANCE: Schizophrenia is a serious psychiatric condition with a worldwide prevalence of approximately 1%. Individuals with schizophrenia experience very severe symptoms and are at an increased risk for suicide, unemployment, permanent disability, and homelessness. Affected adolescents experience even more severe symptoms, tend to be more chronically dysfunctional, suffer from greater cognitive impairments, and may have greater functional and social disability than those with adult-onset schizophrenia. Unfortunately, the cause of schizophrenia is currently unknown. Results of our studies will provide a detailed characterization of brain cell function in patients afflicted with schizophrenia and will offer insight into the mechanisms that contribute to this complex, devastating disease.

DESCRIPTION (provided by applicant): Oligodendrocyte precursors (OPCs) differentiate into oligodendrocytes that are the myelinating cells of the vertebrate CNS. Myelin sheaths wrap axons in the brain and spinal cord and maintain axonal function and promote rapid conduction of electrical impulses. Any damage to myelin sheaths, such as occurs in multiple sclerosis, results in loss of axonal conduction and ultimately axonal degeneration and irreversible neural disability leading to serious physical or mental impairments. Multiple sclerosis is a devastating disease that affects more than 300,000 individuals in the United States. Current therapies are directed towards regulating the inflammatory aspects of the disease, however long term functional recovery will depend upon successful myelin repair in the CNS. Recent studies suggest that many areas of demyelination in the brains of MS patients contain OPCs but the ability of these cells to repair damage is limited because they fail to differentiate for reasons tat are currently unknown. A detailed understanding of the mechanisms controlling OPC maturation and myelination will therefore provide new insights and novel therapeutic strategies for enhancing myelin repair in MS. The experiments described in this proposal will explore the roles of the intracellular signaling molecule cyclin dependent kinase 5 (Cdk5) and its co-activators p35/p39, in regulating the development of OPCs, myelination and remyelination. Cdk5 is known to be involved in various signaling pathways that are key for CNS development. Our preliminary data has revealed novel functions of Cdk5 in controlling the development of OPC and myelination. The proposed study will explore whether Cdk5 within cells of the oligodendrocyte lineage regulates their development and myelination in vitro and in vivo using molecular and genetic approaches. We will identify the roles p35/p39 play in mediating Cdk5 modulation of OPC maturation and myelination. To determine whether the Cdk5 pathway is a novel potential target for therapeutic development we will test whether Cdk5 is essential for remyelination in adult CNS after the induction of focal demyelinating lesions. To accomplish this we will selectively delete Cdk5 from OPCs in the adult CNS during demyelination. Successful completion of the proposed studies will provide critical insights into the signaling mechanisms regulating OPC maturation and myelination and provide novel targets to promote myelin repair. PUBLIC HEALTH RELEVANCE: Multiple sclerosis is a devastating, irreversible and progressive disease that result from failure of myelin repair and degeneration of demyelinated axons. Unfortunately, no effective therapeutics are currently available to promote myelin repair. Understanding OPC differentiation and myelination is critical for developing new strategies for promoting repair of myelin lesions in MS patients. We have identified Cdk5 as an important regulator of oligodendrocyte development and the current studies will define the role of Cdk5 and its activators, p35 and p39 in myelination and myelin repair. Results of our studies will provide critical insights into the signaling mechanisms regulating OPC maturation and myelination and provide novel targets to promote myelin repair.

DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a complex, debilitating neural disorder associated with significant morbidity and mortality. Disease etiology is a result o unknown environmental and genetic factors, while disease pathology presents as inflammatory injury to the central nervous system (CNS) causing physical incapacity. This damage is a result of autoimmune-mediated destruction of oligodendrocytes causing demyelination. Prolonged demyelination leads to axonal damage occurring either in focal lesion sites and/or widespread throughout the CNS, depending on presentation of the disease. MS is treated with disease-modifying agents that target the immune system in an attempt to reduce the frequency of relapses and delay disease progression. These drugs have had limited success in improving patient outcomes and lack the unmet need to promote immediate remyelination. Identifying drugs that induce resident oligodendrocyte progenitor cell (OPC) mediated remyelination would provide patients a vital mechanism to halt disease progression and improve function. We propose to screen drugs that promote remyelination for repurposing in MS. To achieve this goal we have developed a proposal that incorporates the skill and expertise of multiple disciplines through a proven validation pipeline to interrogate drug entities that promote remyelination. We will begin with drugs that have a history of use in human clinical trials and assess their ability o promote rodent OPC maturation and myelin gene expression in our novel high throughput in vitro system. This system takes advantage of our technology to generate scalable sources of functional OPCs from pluripotent stem cells. Validated drug hits from our high throughput in vitro screen will then be tested in a tractable and economical ex vivo myelination assay utilizing organotypic rodent brain slice cultures. Moving forward, we will take our 5 most promising drug hits (as determined from in vitro and ex vivo assays) and test them in animal model systems regularly used to interrogate applicability to MS (lysolecithin-mediated spinal cord demyelination and Experimental Autoimmune Encephalomyelitis). Top drug hits will also be tested on human OPCs. Using our strengths in preclinical MS research and stem cell differentiation, we aim to identify efficacious pharmacological agents that promote remyelination by the end of this two year project. We are prepared to progress lead drugs identified from our studies to human clinical trials.

? DESCRIPTION (provided by applicant): Multiple sclerosis (MS) is a complex, debilitating neural disorder associated with significant morbidity and mortality. Disease etiology is a result o unknown environmental and genetic factors, while disease pathology presents as inflammatory injury to the central nervous system (CNS) causing physical incapacity. This damage is a result of autoimmune-mediated destruction of oligodendrocytes causing demyelination. Prolonged demyelination leads to axonal damage occurring either in focal lesion sites and/or widespread throughout the CNS, depending on presentation of the disease. MS is treated with disease-modifying agents that target the immune system in an attempt to reduce the frequency of relapses and delay disease progression. These drugs have had limited success in improving patient outcomes and lack the unmet need to promote immediate remyelination. Identifying drugs that induce resident oligodendrocyte progenitor cell (OPC) mediated remyelination would provide patients a vital mechanism to halt disease progression and improve function. Using an innovative pluripotent stem cell-based high throughput screening platform we have discovered a class of FDA approved drugs that enhance remyelination in mouse models of MS. In this grant we will use in vivo and in vitro models to study the cellular and molecular effects of this class o drugs on mouse and human OPCs. These studies will provide the basis for potential translation of this drug, or modified derivatives, to clinical testing as a remyelinating therapeutic.

Project Abstract Although modern therapies have dramatically improved the outlooks for people living with HIV/AIDS (PLWHA) they are unable to cure infection, leaving these individuals burdened by a lifelong commitment to expensive antiretroviral medication. It has also become clear that these treatments do not fully restore health, nor do they address the negative social issues associated with being HIV positive, including stigma and issues related to criminalization. The development of a safe and effective HIV cure would thus greatly improve the lives of PLWHA. A major obstacle to curing HIV infection is the establishment of reservoirs of hidden or `latent' virus which evade the immune system and can re-seed infection if an individual stops antiretroviral therapy. Considerable efforts are underway to attempt to purge these HIV reservoirs in the blood and in other tissues in the body where the virus is known to persist. However, an important unknown currently hangs over these efforts. HIV is known to be able to infect certain cells in the brain and it is presently not known whether or not this virus can persist for long periods of time in antiretroviral-treated individuals and re-establish spreading infection. Since many of the therapies currently being tested to eliminate HIV do not target virus in the brain, this raises the possibility that even treatments that are very effective in the blood and other tissues may not cure infection. In this study we propose using a novel mouse model of HIV infection to first determine whether or not HIV can be passed from the brain to the blood and other tissues, and then to test how long any infectious virus in the brain can persist. If the virus in the brain persists for only short period of time then it is possible that sustained antiretroviral therapy will be sufficient to eliminate this reservoir in PLWHA, however if it is more durable then other types of therapies will have to be developed. In thus study we will also test two such therapies by determining whether antibodies or killer T cells that target HIV can prevent the spread of virus from the brain, either by entering into the brain and eliminating HIV at this source, or by eliminating any `beachheads' of virus in the blood or tissues from HIV that exits the brain. Thus, our study will both lead to a better understanding of the challenge that HIV reservoirs in the brain may pose to curing HIV infection, and will test therapeutics with the potential to overcome this challenge and contribute to curing infection.

Project Summary This resubmission proposal aims to elucidate the role of a histone deacetylase, HDAC11, in diseases such as multiple sclerosis (MS), and to establish HDAC11 inhibition as a potentially effective new treatment strategy for diseases including MS. MS is a chronic, immune-mediated demyelinating disease of the central nervous system. Like many autoimmune disorders, it presently has no known cure, and current drugs available for managing this disease are only effective early on and are accompanied by many adverse effects. The disease mechanism of MS remains unclear, and no effective targeted therapy is available for chronic progressive MS. Our preliminary studies show that deletion of HDAC11 ameliorates clinical symptoms in a mouse model of MS. In parallel, we discovered a novel HDAC11 enzymatic activity that is >10,000-fold more efficient than its deacetylase activity. This novel activity allows us to begin to uncover physiologic substrates of HDAC11, which in turn will help to uncover the biological mechanisms of HDAC11?s actions. One of the goals of this research is to investigate how this newly discovered enzymatic activity underlies the immune-regulatory function of HDAC11 in MS. Knowledge gained from these studies will help to further understand the disease mechanism of MS and to develop better therapeutics. Because the discovery of a novel HDAC11 activity has enabled us to develop, for the first time, HDAC11-specific inhibitors, the chief objective is to further improve these inhibitors and test whether they can be used to treat diseases such as MS in our established mouse models. Our multidisciplinary team has expertise in all aspects needed to make this project successful. Overall, the proposed studies in this application will not only yield a better understanding of HDAC11?s function in health and diseases, but may also result in a first prototype targeted therapy for the treatment of chronic progressive MS, and possibly other diseases as well.