Yanming Wang, Ph.D. - US grants

DESCRIPTION (provided by applicant): In this application for a Mentored Quantitative Research Career Development Award (K25), the candidate's research and career development plans are described. The project is designed to customize the educational and research activities for the candidate to achieve two major goals. The immediate goal is for the candidate to continue his research in amyloid imaging in Alzheimer's disease and aging. The long-term goal is for the candidate to acquire advanced biomedical research skills and develop as an independent researcher in aging-related biomedical imaging. To achieve these goals, the candidate will obtain further trainings in neuroscience, biostatistics, pharmacology, and pharmacokinetics as well as in responsible conduct of biomedical and clinical research. He will also acquire related knowledge through journal clubs, research seminars, lectures, and conferences, and through interaction with other investigators and trainees. The practical skills in biomedical imaging will primarily be obtained through the proposed microPET studies under the guidance of Drs. Mathis and Klunk at the University of Pittsburgh. In this proposed research, the candidate plans to use microPET to evaluate amyloid-imaging agents in transgenic mice models of amyloid deposition. This will allow us for the first time to evaluate the in vivo binding specificity and pharmacokinetic profiles of lead compounds in a CNS model that mimics the future human studies. Therefore, this project will satisfy the following specific aims: 1) rationally design and synthesize a selected array of amyloid-binding agents; 2) evaluate the new compounds for in vitro binding affinity and specificity for amyloid deposits; 3) evaluate selected compounds in ex vivo studies of brain entry, crearance; and metabolism in normal control mice with no amyloid deposits in the brain; 4) use microPET to assess the in vivo properties of selected compounds in amyloid-containing transgenic mouse models to determine in vivo binding specificity and detailed pharmacokinetic profiles. The overall goal of our research is to identify potent, selective, and brain permeable amyloid probes suitable for in vivo human studies.

[unreadable] DESCRIPTION (provided by applicant): The proposed research is directed at developing small-molecule probes that readily enter the brain and specifically bind to myelin membranes. Abnormality and changes associated with myelin are seen in many neurodegenerative disorders. Thus, direct assessment of the myelin content in vivo in the central nervous system has been an important goal in protection and repair of axonal damage. Although magnetic resonance imaging (MRI) is conventionally used for detection of brain lesions with high sensitivity, MRI does not directly monitor myelin content. It does not differentiate between lesions caused by demyelination and inflammation. For in vivo MR studies on myelin, molecular probes are required as contrast agents that are specific for myelin membranes. We propose to develop molecular probes suitable for magnetic resonance imaging studies of myelin. Development of myelin-imaging probes would also allow other MR imaging techniques such as diffusion tensor imaging (DTI) and magnetic resonance spectroscopy (MRS) to better studies myelin changes in the brain. We hypothesize that small-molecule probes can be developed as myelin-imaging agents that selectively accumulate in the myelinated brain regions with suitable in vitro and in vivo properties for MR studies. To test this hypothesis, we have identified a lead compound, termed BMB, which readily enters the brain and selectively binds to myelin. We plan to further evaluate this lead myelin-imaging agent to address the following specific aims: 1) Further evaluate the lead myelin-binding compound through in vitro binding assays and tissue staining to quantitatively determine the binding affinity and specificity; 2) Characterize the in vitro MR properties of BMB following binding to isolated brain myelin fractions and incubation in brain tissue sections; 3) Characterize the in vivo MR properties of the probes in normal control mice and mouse models of demyelination and remyelination. It is anticipated that completion of the project will prove the concept that molecular probes can be developed for in vivo MR studies of myelin, and serve as a basis for further development and application in human subjects. This research has following impacts on public health: 1) effective detection of demyelination at early stages to aid in definitive diagnosis; 2) correlation of the demyelinated lesion burden with the severity of symptoms, and 3) facilitation of efficacy evaluation of remyelination therapies currently under development. [unreadable] [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The proposed research is aimed at developing radiopharmaceuticals that readily enter the brain and selectively localize in the white matter via direct binding to myelin membranes. Abnormality and changes associated with myelin membranes in the central nervous system play a key role in the pathogenesis of multiple sclerosis and other related neurodegenerative disorders. Thus, direct assessment of myelin content in vivo has been an important goal in protection and repair of axonal damage. However, the lack of molecular probes has limited the progress of myelin imaging and hindered efficacy evaluation of novel myelin repair therapies currently under development. To meet this need, we plan to develop myelin-imaging agents uniquely suited for use in clinical imaging modalities such as positron emission tomography (PET). We hypothesize that small-molecule PET probes can be developed, which will freely enter the brain, directly and selectively bind to myelin membranes. To test this hypothesis, we have identified some lead compounds with promising binding properties for in vivo studies, which allows us to address the following specific aims: 1) Rationally design and synthesize a selected series of myelin-imaging agents and quantitatively evaluate their binding properties in vitro for structure-activity relationship studies;2) Radiolabel selected agents with positron-emitting 11C or 18F and assess the in vivo binding properties in animal models of demyelination to determine the rates of brain entry, clearance, and specific retention in the brain;3) Evaluate the pharmacokinetic profiles and metabolism of selected myelin-imaging agents through PET studies in non-human primates for potential application in human subjects. It is anticipated that completion of the project will lead to the development of imaging markers that are suitable for application in human subjects. PUBLIC HEALTH RELEVANCE: This research project has following important impacts on public health: 1) facilitation of efficacy evaluation of therapies currently under development that are aimed at prevention of axonal damage and myelin repair;2) direct correlation of myelin changes with clinical outcomes;3) aid in early and accurate diagnosis and subtyping of MS and related diseases.

DESCRIPTION (provided by applicant): Post-translational histone modifications play important roles in chromatin functions, ranging from DNA damage and repair, DNA recombination, chromatin structure, to gene regulation. p53 is a tumor suppressor and transcription factor that recruits both coactivators (e.g., histone acetyltransferases and protein Arg methyltransferases) and corepressors (e.g., histone deacetylases) to its target gene promoters to regulate chromatin structure and gene expression. In addition, the activity of p53 as a tumor suppressor and transcriptional factor is also regulated by numerous post-translational modifications of p53 itself, including phosphorylation, acetylation, and ubiquitination. Histone deacetylases (HDACs) counteract the activity of histone acetyltransferases (HATs) to dynamically regulate histone acetylation and finely adjust the expression of p53 target genes. Although protein Arg methyltransferases (PRMTs) have been found to methylate histone Arg residues to activate p53 target genes, the process to reverse histone Arg methylation is unclear. I have reported that peptidylarginine deiminase 4 (PAD4) can reverse histone Arg methylation via a reaction called demethylimination thereby repressing the estrogen receptor target genes. Recent studies from my group have showed that PAD4 works as a p53 corepressor to counteract the activities of PRMTs and to reverse histone Arg methylation at the p53 target gene p21 promoter. In addition, our preliminary studies demonstrated protein-protein interactions of p53/PAD4/HDAC2. The central hypothesis to be tested in this proposal is that citrullination and deacetylation of histones and p53 regulate p21 expression by overlapping molecular mechanisms. To test this hypothesis, we will 1) characterize nucleosome positioning/density of the p21 promoter and relate the change of chromatin structure with the activation of p21 (Aim 1);2) investigate how PAD4 and HDAC2 cooperate to efficiently repress the expression of p21;3) analyze whether reversible p53 acetylation catalyzed by HAT/HDAC forms a molecular switch to control the p53/PAD4 interaction;4) investigate the effects of p53 citrullination on the p53 activity in DNA binding and gene regulation. PUBLIC HEALTH RELEVANCE: p53 is mutated in about half of human cancers and plays a pivotal role in the cellular response to cope with various stresses, including DNA damage and hypoxia. The role of PAD4 as a p53 corepressor implicates that one can increase the expression of the p53 target genes by blocking the activity of PAD4. Consistent with this idea, inhibition of PAD4 by its inhibitor or depletion of PAD4 by its siRNAs increased the expression of the p53 target gene p21, suggesting that PAD4 is a hopeful novel target for cancer treatment.

ABSTRACT Multiple sclerosis (MS) is an autoimmune demyelinating disease characterized by myelin damage in the brain and spinal cord. The current diagnosis and management of MS rely primarily on magnetic resonance imaging (MRI), which provides a means to detect overall changes in tissue water content. However, lesions detected by MRI reflect only macroscopic tissue injuries that are not necessarily caused by myelin damage. Consequently, the use of MRI as a primary measure of disease activity is poorly correlated with clinical outcomes in MS. This long-standing clinico-radiological paradox in MS is considered as a missing link to finding a cure for MS as it hampers efficacy evaluation of putative MS therapies, particularly myelin-repair therapies that are designed to promote long-term functional restoration. To overcome this challenge, we hypothesize that positron emission tomography (PET) imaging, when used in combination with myelin-specific radiotracers, will be able to directly detect and quantify changes of myelin distribution in the brain and spinal cord and that measurement will correlate with clinical evaluation. To test this hypothesis, we have developed a series of myelin-imaging agents that readily penetrate the blood-brain barrier and selectively localize in the brain and spinal cord in proportion to the myelin content. In preliminary studies, we identified a lead radioligand, termed [11C]MeDAS, that is specific for PET imaging of myelin changes and suitable for translational studies. In order to implement [11C]MeDAS-PET in a clinical setting, we plan to address the following specific aims: 1) Characterization of the binding properties of MeDAS in the postmortem human brain and spinal cord tissues; 2) Conduct [11C]MeDAS- PET imaging in non-human primates; and 3) Conduct Phase I/II studies in human subjects to evaluate safety and provide initial proof-of-concept data for measurement of myelin content. Successful completion of these studies will validate [11C]MeDAS-PET as a unique imaging marker for unambiguous monitoring of disease progression or recession and myelin-repair processes in MS. !

Abstract Current diagnosis, prognosis, and management of multiple sclerosis and related neurological disorders primarily rely on magnetic resonance imaging (MRI), which is widely used in selecting patients for immune- modulatory treatment, monitoring disease activity, and predicting treatment response. However, MRI is limited to be a measure of overall changes in tissue water content and reflects only macroscopic tissue injuries, which may be caused by a combination of pathological activities ranging from edema and inflammation to demyelination and axonal loss as observed in MS. Without any specificity for myelination, the use of MRI as a primary measure of disease activity is found to poorly correlate with clinical outcomes in MS. This long- standing clinico-radiological paradox in MS is considered as a missing link to finding a cure for MS as it hampers efficacy evaluation of putative MS therapies, particularly myelin-repair therapies that are designed to promote long-term functional restoration. To overcome this clinico-radiological paradox in MS, we hypothesize that positron emission tomography (PET) imaging, when used in combination with myelin-specific radiotracers, will be able to directly detect and quantify changes of myelin distribution in the brain and spinal cord and that measurement will correlate with clinical outcomes. To date, we have identified two radiotracers for PET imaging that complement each other in detection and quantification demyelination and remyelination: one is myelin-targeted [11C]MeDAS that is designed to monitor global myelin distribution and the other is potassium- channel targeted [18F]3-F-4-AP that is designed to monitor exposure of axons after myelin damage. Given each radiotracer alone will not be able to provide ubiquitous information, we plan to combine the two radiotracers and determine their synergistic utility for accurate detection and quantification of demyelination and remyelination in different animal models of MS. In this project, the following specific aims will be addressed: Aim 1. Conduct sequential microPET imaging using [11C]MeDAS followed by [18F]3-F-4-AP in a LPC rat model and correlate the imaging results with demyelination and remyelination; Aim 2. Conduct sequential microPET imaging using [11C]MeDAS followed by [18F]3-F-4-AP in an EAE rat model. After immunization, the same animals will be scanned at various time points to determine the pharmacokinetics of each tracer in the spinal cord. Aim 3. Determine the time courses of demyelination and remyelination through cross-reference of the longitudinal MeDAS-PET and [18F]3-F-4-AP-PET scans in the same animals. Completion of this project will deepen our understanding of the disease mechanisms by accurately monitoring the time course of demyelination and/or remyelination in vivo and develop parametrics for direct correlation with functional recovery and efficacy evaluation of new drug discovery and development.