Lu Lu - US grants

DESCRIPTION (provided by applicant): Alcoholism is a complex multi-factorial disease with a strong genetic component. The etiology of this disease is due in part to genetic differences in responses to stressors and to variation in the efficacy of ethanol in stress reduction. The molecular basis of this interaction will be studied, leading to a direct evaluation of the hypothesis that mechanisms of stress mediation by alcohol share common molecular pathways with stressors. It will be possible to determine the degree to which shared and separate mechanisms are employed. The project exploits a recently developed microarray technology called transcriptome-QTL mapping to identify the sources of variation in gene expression in the mouse hippocampus. This method enables global mapping of factors controlling the transcriptional response to stress and its modification by alcohol. A large panel of Long Sleep by Short Sleep (LXS) recombinant inbred (RI) strains of mice with sufficient power for the study of traits with small effect or low heritability will be used to map the regulatory loci revolved m mediating the infraction between ethanol and stress. Stressed and unstressed animals will be treated with ethanol or left untreated. Behavioral and physiological stress phenotypes will be measured and mapped in all RI strains. Using oligonucleotide arrays, all strains of the RI panel will be assessed for stress and ethanol related gene expression differences. In the final stage of this work, the identification of the genetic basis of expression differences will be carried out using the novel, large-scale transcriptional analysis to identify major regulatory elements and gene networks that influence the molecular, physiological and behavioral manifestations of alcohol and stress response. This project will generate publicly available transcriptome regulation resources for the alcohol and neuroscience research communities. Because this project focuses on naturally occurring genetic variation in the mouse, the results are likely to extend to human populations with differences in the stress-alcohol interaction.

DESCRIPTION (provided by applicant): In this proposal, we use our enlarged set of BXD recombinant inbred strains to identify gene loci that are involved in modulating the severity of glaucoma. Our approach combines a thorough clinical and laboratory examination, and microarray analysis of the entire set of 81 BXD lines generated over the last 10 years with the express purpose of studying the genetics of eye disease and glaucoma. One of the parental strains of BXD, DBA/2J, develop an age-related glaucoma that is preceded by iris atrophy and pigment dispersion. While mutant alleles of two genes, Tryp1 and Gpnmb, cause the iris disease in D2, the literature strongly suggests that these mutations are not sufficient to cause glaucoma. Specifically, introgression of both mutant alleles onto C57BL/6J, the other parent strain of BXD, results in a marked resistance to optic nerve damage. This indicates that genes other than those that cause pigment dispersion influence the glaucomatous phenotype. The current proposal describes a unique opportunity to define the modifying loci. In Aim 1, we test the hypothesis that the combined mutations in Tryp1 and Gpnmb are not sufficient to cause all aspects of the glaucoma phenotype. If our hypothesis is true, we expect to see that the severity of disease is not solely dependent on the two mutant alleles. We have already identified multiple BXD strains in which IOP and genetic diplotype are not correlated. As we systematically examine all BXD strains and establish relations between phenotype and diplotype, we fully expect to find additional strains that defy expectations of a simple two-locus disease model. In Aim 2, we define loci and genes that modulate glaucoma severity. To do so, we will identify and evaluate candidate genes within loci that modulate the severity of the glaucoma phenotype. We will exploit our new whole genome shotgun sequence for D2 (about >50x short read coverage generated at UTHSC in 2009) along with massive whole eye and whole retina expression datasets that we have also generated as a prelude to this work. We expect to efficiently nominate and evaluate candidate glaucoma genes using state-of-the-art bioinformatic methods and conventional molecular assays. In Aim 3, we use bidirectional translation. We test the translational validity of mouse candidates from Aim 2 using cohorts of human glaucoma patients. Dr. J. Wiggs and colleagues will perform focused gene association studies using candidate glaucoma genes nominated in Aim 2. Specifically, we use association analyses of markers encompassing syntenic regions of human chromosomes. In reciprocal reverse translation, we (MMJ and LL) will evaluate known, new, and candidate glaucoma genes from clinical cohorts and determine if and how these variants are associated with glaucoma- associated traits in BXDs. Combining the top priority gene candidates from both mouse and human glaucoma studies, we will generate molecular and statistical models of susceptibility candidate genes, linked phenotypes, and associated mechanisms. PUBLIC HEALTH RELEVANCE: Glaucoma is a highly prevalent group of diseases that, if uncontrolled, causes irreversible loss of vision. The underlying cause of the disease is not known in the majority of cases, therefore treatment options are limited to lowering the intraocular pressure. Outcomes of our proposed investigations will identify gene loci that modulate glaucoma severity to expand this sparse list of genes that are known to contribute to this disease.

DESCRIPTION (provided by applicant): Since the discovery of different typologies for alcohol-use disorders in the 1980s, two major patterns have emerged. One type is oftentimes called alcohol abuse, whereas the other is associated with alcohol dependence. The abuse typology is associated with antisocial behavior and has an estimated high heritability, whereas the dependence- based typology is associated with stressful life events, bad marriage, difficult job, etc. This latter type is also supposed to have some degree of heritability, but this is difficult t assess because not all susceptible genotypes are subjected to stressful events. Attempts to develop animal models of the stress-related disorder have yielded inconsistent results. This is because the stressors tend to be short in duration and not always similar to human life events; or, the measure of alcohol drinking may not capture the kind of consumption seen in humans prior, during or following the stressful events. The primary aim of this proposal is to develop a model of stress-related change (increase or decrease) in alcohol consumption in a genetic reference population of mice subjected to several weeks of unpredictable environmental perturbations, termed chronic mild stress (CMS). The approach is a systems biology/systems genetics analysis of alcohol consumption within the framework of other physiological changes caused by CMS. Alcohol consumption will be assessed by the drinking in the dark (DID) paradigm and will be measured prior to CMS, during CMS and following CMS. The physiological measures include hypothalamus-pituitary-adrenal axis function, i.e., fecal corticosterone determinations during all phases of the experiment, thymus and adrenal weights. All endpoints will be subjected to multivariate analysis and genetic analysis to identify polymorphic genes that influence alcohol drinking and the other indices. Gene expression by microarray analysis will be performed on hippocampus, hypothalamus and adrenal glands to identify genes whose expression is altered by CMS and which genes that change expression are related to the other parameters, especially DID. At the end of the work, we will identify genes and gene networks related to stress-related alcohol consumption and that are syntenic with the human genome.

DESCRIPTION (provided by applicant): There are at least two types of Parkinson's disease (PD), familial and sporadic (sPD). By far, sPD accounts for the majority of cases and is becoming to be seen as the result of several genes and their interaction with the environment, including widely used pesticides. One such agent is paraquat (PQ), an herbicide used widely in developing countries and also in the USA. The data from epidemiological studies linking PQ exposure with sPD are inconclusive and we will show that PQ exposure alone is likely insufficient to produce sPD. At least one other factor is iron content [Fe] in the substantia nigra pars compacta (SNc). Iron in the SNc is considered to be another risk factor for sPD. Studies conducted in vitro have shown that PQ and Fe act synergistically in killing dopamine neurons in the SNc, the pathological hallmark of PD. In the proposed research, we will show that PQ disrupts iron homeostasis in the SNc and that the increased iron in this tissue is what defines PQ neurotoxicity. The overall goal of this research is to identify genes and gene networks that confer differential susceptibility to PQ-induced increased Fe in the SNc. In order to address the problem, we will study the effect of PQ- increased Fe in 40 recombinant inbred strains derived from C57BL/6 and DBA/2 parental strains. The first experiment will be to show wide, genetic-based variability in paraquat- increased Fe in the SNc. The second experiment will be to show that PQ-based destruction of dopamine neurons is related to the extent of PQ-related disruption of Fe homeostasis in the SNc. We will next investigate the effects of paraquat on gene expression by microarray analysis in the substantia nigra, pars compacta and then by combining QTL analysis for the gene expression with QTL for PQ-increased Fe in the SNc, we will elucidate the biochemical pathways involved in paraquat-iron neurotoxicity as well as elucidating genetic markers that indicate increased (decreased) risk for damage to dopamine neurons in the SNc

Variability in hypertrophic cardiomyopathy (HCM) clinical manifestation is primarily determined by modifier genes, which are poorly understood. Discovering of the genetic basis of differential vulnerability is critical in predictive and personalized care for patients with HCM and will enabling more comprehensive genetic and genomic screening with an aim to intervene as early as possible and eliminate risk of sudden death. The discovery of modifier genes that contribute to variation and incomplete penetrance of HCM has proven difficult in human cohorts. Large murine genetic reference populations (GRPs) now finally provide a effective solution. The BXD family of strains?currently the largest and best characterized mouse GRP?is made up of 160 highly diverse lines that descend from crosses between C57BL/6J (B6) and DBA/2J (D2) parental strains. The BXDs have been bred specifically for systems genetics studies using both classic forward genetic methods and for reverse genetic studies. We have shown that the D2?father of the BXD cross?is an excellent murine HCM model. D2 contains mutations of Mybpc3 and Myh7, the major causal genes of HCM and the key features of human HCM. In contrast, the B6 (mother of the BXDs) has wild type alleles and normal hearts. The objective of our proposal is to identify modifier genes that affect the severity of HCM phenotypes. Our hypothesis is that interactions of modifier and causal genes govern HCM severity and related phenotypes. The research here involves multi-scale genetic, transcriptomic, molecular and cellular profiling of B6, D2, and up to 100 BXDs. This work will be transformative and lead to the identification of strong candidate genes and networks underlying individual differences in HCM phenotypes. Aim 1: Systematically quantify HCM-associated traits and their variability and heritability across 100 BXD genotypes of isogenic mice. The purpose of Aim 1 is to determine the clinical, laboratory and molecular HCM phenotypes in 100 BXD strains, setting the stage for us to explore genetic variation, cofactors, and mechanisms of HCM in Aims 2 and 3. Aim 2: Define genes that modulate the severity of HCM. Building upon the phenotype data generated in Aim 1, as well as the already acquired sequence and transcriptome data for B6 and D2, and BXDs, we will identify strong gene variants that modulate variability of HCM phenotypes using state-of-the-art system genetic strategies and conventional molecular and cellular assays. Aim 3: Test the translational validity of mouse HCM modifier gene candidates. We will justify candidate genes identified in Aim 2 with established HCM human GWAS data. In reciprocal reverse translation, we will evaluate candidate HCM genes from human cohorts and determine whether these variants are associated with HCM-associated traits in BXDs. Combining the top priority gene candidates from both mouse and human HCM studies, we will generate molecular and statistical models of susceptible candidate genes, linked phenotypes, and relevant mechanisms. We will finally validate genes modulating HCM phenotype severity using loss-off function strategy.

This Major Research Instrumentation grant will develop a new experimental exoskeleton for rehabilitation that will stimulate a wide range of research studies to improve the lives of individuals who have difficulty walking due to a stroke, spinal cord injury or cerebral palsy. Studies have shown that individuals with ambulation disabilities express a preference for being able to walk independently in their communities. Robotic exoskeletons that have motors to assist with leg joint movement have sparked great interest, but have not yet been able to provide the quality of walking that meets user needs. Walking is robotic and slow, and only works on very flat surfaces. These devices do not yet work in the real world.

This grant will allow New Jersey Institute of Technology to develop a research-quality exoskeleton that will be used in more than twelve projects aimed at improving the ability of exoskeletons to provide assistance in the home and community. The new exoskeleton will have powerful motors to support all major movements of the human leg, as well as a large array of sensors. It will have the computer power to explore exciting new approaches to both user and automatic control of walking and balance. The projects will address important engineering and scientific questions about human-robot interaction, and will produce more effective user-robot interfaces that are intuitive, easy to learn, and allow the user to keep his/her balance. This research will help define the capabilities that are essential in a commercial exoskeleton and will the next generation of commercial products. This work supports the mission of the NSF by providing essential technological study of robot controls, robot sensors and computational methods, human performance, and the neuroscience of walking. This science is essential to promoting further advances in neurorehabilitation services and rehabilitation product development. In addition, its presence in a major technological university will have a major impact on the education of graduate and undergraduate students, as well as bringing technological excitement to a large number of secondary school students.

This Small Business Innovation Research Phase I project aims to provide an affordable sealant for sealing expansion joints and cracks in concrete pavement, bridge deck, etc. In transportation infrastructure, expansion joints are intentionally constructed in order to allow movement of the structural elements due to linear thermal expansion when temperature rises. In addition, cracks are a common failure mode in pavement. If they are not properly sealed, water penetration will damage the surface layer and the layers beneath, and entrapped debris will cause rupture of the concrete wall. Therefore, sealing cracks and joints is a common practice to maintain or extend the structure service life. Various types of sealants have been used with an annual market value about $6.1 billion. Unfortunately, many sealants cannot properly seal cracks and joints, and/or last long, requiring frequent replacement or resealing. In this project, a smart sealant that expands upon cooling and contracts upon heating, which is thermally opposite to concrete, will be developed to counteract thermal movement of the joined structural elements.

The intellectual merit of this project lies in the feasibility of a smart sealant technology. The primary reason for joint failure is that most sealants behave similar to concrete, i.e., they contract upon cooling and expand upon heating. This thermal behavior is contrary to the requirement for sealants. The objective of this project is to design, synthesize, characterize, and evaluate a cost-effective two-way shape memory polymer based sealant for sealing expansion joints or cracks in concrete pavement or bridge deck, which will expand upon cooling and contract upon heating. It will have the required mechanical properties and durability to survive the repeated traffic load and outdoor environment. The success of the project can have beneficial impact not only on the transportation infrastructure but also other structures such as driveways, parking lots, dams, harbors, buildings, swimming pools, etc.

There are at least two types of Parkinson's disease (PD), familial and sporadic (sPD). By far, sPD accounts for the majority of cases and is becoming to be seen as the result of several genes and their interaction with the environment, including widely used pesticides. One such agent is paraquat (PQ), an herbicide used widely in developing countries and also in the USA. The data from epidemiological studies linking PQ exposure with sPD are inconclusive and we will show that PQ exposure alone is likely insufficient to produce sPD. At least one other factor is iron content [Fe] in the substantia nigra, pars compacta (SNc). Iron in the SNc is considered to be another risk factor for sPD. Studies conducted in vitro have shown that PQ and Fe act synergistically in killing dopamine neurons in the SNc, the pathological hallmark of PD. In the proposed research, we will show that PQ disrupts iron homeostasis in the SNc and that the increased iron in this tissue is what defines PQ neurotoxicity.The overall goal of this research is to identify genes and gene networks that confer differential susceptibility to PQ-induced increased Fe in the SNc. In order to address the problem, we will study the effect of PQ-increased Fe in 40 recombinant inbred strains derived from C57BL/6 and DBA/2 parental strains. The first experiment will be to show wide, genetic-based variability in paraquat-increased Fe in the SNc. The second experiment will be to show that PQ-based destruction of dopamine neurons is related to the extent of PQ-related disruption of Fe homeostasis in the SNc. We will next investigate the effects of paraquat on gene expression by microarray analysis in the substantia nigra, pars compacta and then by combining QTL analysis for the gene expression with QTL for PQ-increased Fe in the SNc, we will elucidate the biochemical pathways involved in paraquat-iron neurotoxicity as well as elucidating genetic markers that indicate increased(decreased) risk for damage to dopamine neurons in the SNc

Abstract Cardiomyopathies (CMs) are group of inherited heterogeneous diseases of heart muscle with no definite effective treatment, ultimately resulting in heart failure (HF), transplant or sudden cardiac death (SCD) in many patients. Despite decades of intense research, it is still difficult to predict CM phenotypes and explain clinical heterogeneity, severity and prognosis. The likely reason for poor genotype-phenotype association is that mutations in single gene do not completely determine disease course; rather interactions of multiple genes, causal and modifier, may be required to explain CM phenotypes. We have screened adult and pediatric patients with various types of CMs, including dilated (DCM), hypertrophic (HCM) and restrictive (RCM) using whole exome sequencing (641 patients) and direct Sanger sequencing (900 patients), and identified myopalladin (MYPN), encoding a cytoskeletal Z-disk protein, as a strong causal gene associated with heterogeneous CM phenotypes with clinical expressions ranging from asymptomatic left ventricular hypertrophy to dilation with progressive HF to SCD or transplant. The CM symptoms are highly varied among individual patients, even within the same family members who carry identical mutations. These variations are influenced by modifier genes that alter the effect of causal gene at major locus. However, modifier genes of MYPN remain largely unidentified and are likely to depend on the interaction of multiple genes in MYPN related gene pathways and gene networks. The identification of modifier genes is now a crucial goal of research in CMs from the viewpoints of diagnosis, treatment and genetic counseling, but it remains very challenging in clinical cohorts. The objective of the current study is to determine modifier genes and molecular networks that modulate severity of MYPN induced CMs using powers of combined reverse and forward genetics and systems genetic analysis. Systems genetics is such an approach to understand complex diseases by focusing on how genes work together in groups rather than singly. We have confirmed that mutation Q529X of MYPN associated with heterogeneous phenotypes in humans causes CM in knock-in mice. We have developed the largest and best characterized mouse genetic reference population (GRP) composed of over 150 lines derived from crosses between C57BL/6J (B6) and DBA/2J (D2) parents. The D2 strain has been identified as mouse CM model, and CM phenotypes from D2 mouse is segregated among the BXD family of strains, which makes BXD family as an excellent platform to identify CM modifiers. Moreover, we have introduced Q526X-Mypn mutation (homologous to human Q529X-MYPN) into BXD genetic background to examine how different genetic background modifies the effect of Mypn mutation on CM phenotypes. This proposal is one of the first multidisciplinary collaborative efforts to identify modifier genes in MYPN induced CM in both human and mouse. Using cross-species validation sources and powerful systems genetics, we will define and validate novel modifier genes that interact with Mypn and are responsible for CM variations.

Metabolic syndrome (MetS) is a complex trait characterized by multiple abnormalities in glucose and fat metabolism, involving incompletely understood biological networks between various organs, and influenced by genetic and environmental (GXE) interactions. GPRC6A is a nutrient sensing G-protein coupled receptor implicated in the unique regulation of energy metabolism. In genetically engineered mouse models (GEMMs), GPRC6A regulates glucose and fat metabolism and prevents high fat diet (HFD) induced metabolic complications through direct tissue-specific effects and the release of hormones that coordinate metabolic functions between organs. The complexity of the cellular and systemic metabolic networks regulated by GPRC6A, the variable phenotypes in GEMMs, and the limited understanding of GPRC6A functions in humans are critical barriers to defining the role of GPRC6A in preventing and treating MetS and its complications. Our central hypothesis is that GXE interactions influence GPRC6A regulation of energy homeostasis. Aim 1 will test the hypothesis that GXE inter- actions modify GPRC6A regulation of glucose and fat metabolism in the liver and other metabolically active organs using GEMMs and a reductionist approach. Experiments will use wild-type GPRC6A-KGRKLP and GPRC6A null mice, HFD, and GPRC6A agonists to explore the effects of HFD and loss- and gain-of GPRC6A function on energy metabolism in mice. The functional significance of the recently evolved human GPRC6A_KGKY genetic polymorphism will be tested in a ?humanized? Gprc6a_KGKY_knockin mouse. We will characterize hepatocyte-specific Gprc6a knockout mice (Gprc6aliver-cko) to investigate GPRC6A?s function in liver, as a prototypic organ controlling glucose and fat metabolism. In Aim 2, we will use groundbreaking resources for systems genetics systems to test hypothesis that genetic backgrounds modify the metabolic effects of GPRC6A and HFD. We will collect metabolic phenotypes and molecular expression data from the livers of BXD recombinant inbred lines treated with a HFD and the GPRC6A agonist, osteocalcin (Ocn). Then we will apply systems biology approaches to define signaling pathways, metabolic processes and gene networks involving GPRC6A regulation of hepatic fat and glucose metabolism. Cell, molecular and mouse genetic approaches will validate these pathways and net- works predicted by systems biology. The predictive power of experimental and computational systems biology approaches to incorporate and integrate distinct levels of information and scientific knowledge of complex systems created by GPRC6A will improve the rigor and reproducibility of preclinical studies of GPRC6A effects on MetS. Our impact will be to: 1) establish the organ-specific functions of GPRC6AKGRKLP and GPRC6AKGKY variants and determine if these polymorphisms alter the susceptibility to and treatment responses of MetS and its metabolic complications; 2) identify the GPRC6A-regulated gene networks controlling glucose and fat metabolism and determine the genetic modifiers that influence the effects of GPRC6A and HFD on MetS; and 3) validate GPRC6A as a unique molecular target for understanding the pathogenesis and treatment of MetS.