Michael C. Wilson - US grants

The goal of this study is to determine the mechanisms governing the tissue specific and coordinate genetic control of the multigene family encoding the polymorphic glycoprotein, gp70. These developmentally regulated proteins, present in specific tissues of normal virus-free mice, are related to the major glycoprotein of the murine leukemia virus. This system, therefore, provides a model for the study of the regulation of cellular gene expression which can be initially approached through the use of homologous viral probes. We will isolate recombinant cDNA clones of the distinct endogenous gp70 mRNAs from viral-free mice and analyze them by restriction mapping and DNA sequencing to document the polymorphic nature of the gp70 protein family. These unique probes will be used to determine whether the restrictive control of tissue specific gene expression and coordinate control of all members of this gene family by a separate regulatory locus are achieved at the level of transcription or by post-transcriptional processing of the mRNA. The genomic DNA bearing the structural genes encoding the individual gp70 proteins will be isolated by recombinant DNA methodoloty. The structure of the cellular genes will be compared to the genome of infectious virus to determine if their restricted expression as gp70 and p30 is the consequence of major structural modification. The gp70 genomic DNA clones will be used to isolate neighboring regions in the DNA to distinguish whether the expression of the gp70 genes is determined by their chromosomal location adjacent to cellular genes. Our long term goal is to identify those sequences in or flanking the structural gene which are the recognition sites of elements controlling gene expression. Specifically, it is important that genes encoding gp70 are potentially of retroviral origin but expressed under cellular control during normal development. Thus, the study of the mechanisms which distinguish the expression of these endogenous genes from infectious virus associated with leukemia will contribute to our understanding of viral oncogenesis.

The goal of this project is to elucidate in molecular terms the mechanisms governing the expression of retroviral transcription units endogenous to the murine genome. Although these retroviral sequences do not result in infectious virus they do encode viral antigens, including a distinct class of the glycoprotein, gp70. Recombination between these sequences and that of infectious ecotropic virus results in the de novo generation of leukemogenic MCF virus in which replacement of ecotropic viral sequence with endogenous information accounts for expanded host range and tissue tropism. Expression of the endogenous retroviral genes is controlled by two distinct, mendelian loci, Gv-1 and Gv-2, requiring positive alleles at both loci for expression of the multiple transcription units. Transcription of individual endogenous retroviral sequences is also independently regulated at the tissue-specific level. The coordinate regulation of endogenous retroviral genes indicates that Gv-1 and Gv-2 encode a trans-acting factor affecting the expression of multiple transcription units. The mechanisms of this gene regulation will be examined in the congeneic partner strains 129Gix+ and Gix-. These strains bear positive alleles at the Gv-2 locus but differ for alternative positive and negative alleles at Gv-1. The genomic DNA containing actively transcribed endogenous retroviral genes will be isolated as recombinant DNA. The cis-acting elements which determine tissue-specific and genetic, Gv-1, responsiveness will be analysed. First, indirect assays of DNase 1 hypersensitive sites in different tissues of Gix+ and Gix- mice will indicate possible binding sites of proteins that confer gene regulation. These studies will distinguish between different transcription units by 5' unique flanking DNA sequence. Second direct analysis of the regulatory sequences will be conducted by measuring the promotor activity in transient transfection assays. Progressive deletion and replacement experiments will locate the position and orientation independent enhancer regions and factor binding sites at the promotor. Finally, strategies are described for identifying and isolating the genetically defined Gv-1 regulatory locus. These studies will lead to further investigation to determine how the Gv-1 product governs transcriptional activity in terms of protein structure and its possible relationship to other eukaryote trans-acting regulatory factors.

The goal is to identify and characterize biochemical parameters that distinguish neuronal cell types within the hippocampal formation. The hippocampus is chosen as a model for cellular and regional specific gene expression because of its relatively simple anatomical organization, consisting of only several major neuronal cell types and because of its well characterized developmental, electrophysiological and neurotransmitter properties. We will examine the anatomical distribution of the expression of specific mRNA transcripts both within the defined subnuclear organization of the hippocampus and with respect to their potential expression in neurons of other regions of the brain. The developmental program of expression will be investigated in both normal and mutant strains of mice to distinguish those transcripts accumulated during cell migration from those expressed only after synaptogenesis. The primary sequence of the encoded protein will be determined and evaluated in terms of potential signal peptides, glycosylation and proteolytic cleavage. These post-translational modifications, together with the neuroanatomical distribution of their expression, should provide important clues as to the nature of the specific proteins. It is believed that these studies will contribute to an understanding of the biochemical basis of hippocampal function. Experimentally induced lesion and clinical studies have associated the hippocampal formation with such complex behavioral phenomena as acquisition and processing of short term memory and propagation of epileptic seizures. For example, a potential animal model for certain forms of epilepsy in humans, the mutant mouse strain "tottering", will be examined for alterations in patterns of gene expression which may result from the primary defect of hyperinnervation by noradrenergic axons of the locus coeruleus to the hippocampal formation. These studies, therefore, could result in future examination of the role of specific gene products during experimentally induced seizure activity.

DESCRIPTION: (applicant's abstract) Proper function of the nervous system requires the development and maintenance of appropriate neuronal connectivity and effective communication that is largely mediated by chemical synapses. Deficits in synaptic connectivity, resulting from genetic or epigenetic abnormalities revealed either during development or after acute pharmacological or environmental insult, are likely to contribute significantly to impaired brain function. Regulated exocytosis of classical neurotransmitters, as well as neuropeptides and modulatory neurotrophic factors is proposed to underlie the necessary presynaptic input for effective communication between neurons. The overall goal of the research of this laboratory is to gain a better understanding of the molecular mechanisms which control neuroexocytosis. Moreover, implicit in these studies is the general hypothesis that presynaptic regulation of neurotransmitter release can modulate brain development and function, and consequently that deficiencies in this molecular machinery can play at least a part in the cognitive and behavioral impairments of neuropsychiatric disorders. To address these issues, we have focused on the protein SNAP-25, which we propose plays a regulatory role, as well as its more well-defined part as an integral structural component of the exocytotic protein machinery necessary for vesicle fusion and neurotransmitter release. Our investigations make use of SNAP-25 null and hypomorphic mouse mutants which we have developed in the previous funding of this grant. In Specific Aim 1 experiments are designed to use Snap25-/- mice to determine the role of neuroexocytosis in axon stabilization and synapse formation. Specific Aim 2 will use replication defective viral expression systems and Snap25-/- cultured neurons to examine role of sequences that are postranslationally modified and distinguish developmentally regulated SNAP-25 isoforms. Specific Aim 3 addresses the hypothesis of whether the developmental switch in SNAP-25 isoforms underlines the maturation of synaptic transmission by electrophysiological and neurochemical measurements of SNAP-25b hypomorph mutants. Finally in Specific Aim 4, experiments are designed to examine whether Snap25 heterozygotes that express reduced levels of SNAP-25 are more susceptible to psychotomimetic drugs and thus are a model of genetic vulnerability. If successful this investigation will shed light on the selective roles of SNAP-25 isoforms, the roles they play in the synaptic plasticity required for normal brain function, and how abnormalities in presynaptic mechanisms of neurotransmission may be involved in neuropsychiatric disease.

This SLC Catalyst activity focuses on individual differences in learning. If individual differences can be known -- and marked somehow in each individual -- then it may be possible to intervene to optimize learning. To fully understand the highly individual learning process, this project aims to resolve in detail the molecular, cellular, and systems basis of patterns of neural activity and networks in rodent models and in humans that contribute to individual learning. The long-range vision is to develop an integrated understanding of individual differences in learning such that it may be possible in the future to tailor teaching methods, or other means of intervention, to an individual based on the function of his/her unique brain system to optimize learning. This Catalyst project plans for a Science of Learning Center through a yearlong activity that emphasizes not only pilot projects to explore the basic research core, but the development of a foundation for diverse disciplines to work together. The research activities proposed have the potential to optimize learning for all individuals of our society. New Mexico as the first minority-majority state is a reflection of the changes occurring throughout the nation and consequently, has an important role as a laboratory for the exploration of the foundations of learning in a highly diverse population. The proposed Southwest Center for the Science of Learning will bring to bear the intellectual and diversity resources to address the complex educational, cultural and economic changes facing the rest of the nation.

This IGERT award supports a broadly-based education and training program at the University of New Mexico that will enable students from various disciplines to develop new nanoscale imaging and sensing tools and to apply them in investigations of fundamental problems in cell biology and neuroscience. By infusing training in cell biology and neuroscience with training in the technological basis of chemistry, electrical engineering, and physics, the university will create an exciting learning environment in which to prepare students for the challenges and opportunities presented by the nanotechnology revolution. The program will include internships at national laboratories and industry, travel to national and international meetings, international collaboration combined with internship placements, and a web-based dissemination. The research component of the program will generate improved understanding of important and relatively unexplored biological processes, enabled by creation of new imaging and sensing instruments. To realize this goal, the program will capitalize on the existing strengths to develop new quantum-dot-based technologies coupled with hyperspectral and multiphoton microscopy that carry the potential to revolutionize research on genomic, cellular, and synaptic activities. The educational component of the program will emphasize collaboration across traditional academic disciplines, with co-advisors from cell biology/neuroscience and engineering/science fields, rotations between various laboratories with different research styles, and active interactions among students with different academic backgrounds. New courses developed under this project will lead to a Graduate Certificate Program in Biomedical Science and Engineering with Concentration in Nanotechnology, to be followed by a new PhD program in Systems Biology and Bioengineering. IGERT is an NSF-wide program intended to meet the challenges of educating U.S. Ph.D. scientists and engineers with the interdisciplinary background, deep knowledge in a chosen discipline, and the technical, professional, and personal skills needed for the career demands of the future. The program is intended to catalyze a cultural change in graduate education by establishing innovative new models for graduate education and training in a fertile environment for collaborative research that transcends traditional disciplinary boundaries.

DESCRIPTION (provided by applicant): There is considerable evidence that the interaction between genetic susceptibly and environmental (GxE) risk factors plays a significant role in neuropsychiatric disease. In particular, GxE interactions are exemplified by attention-deficit hyperactivity disorder (ADHD) and schizophrenia that are prominent heterogeneous mental illnesses, with ADHD affecting 5-8% of school-aged children and schizophrenia having a life-long prevalence of 0.5-1%. While several genetic loci have been implicated in the heritability of these disorders, and environmental factors have been identified from population-based studies, there are few animal models that have been developed to examine the impact of GxE effects to study the mechanism of these interactions. In this application, we address this important goal by exploiting the hypothesis that the convergence of genetically encoded deficits in SNAP-25 expression, resulting selective deficiencies in synaptic transmission, and the environmental impact of prenatal nicotine exposure during brain development leads to emergent alterations of synaptic plasticity in the striatum. The potential involvement of the neural SNARE protein SNAP-25 as a candidate gene for neuropsychiatric disease has been revealed in our early studies of the hyperactive mouse mutant coloboma, more recently by genetic linkage studies in the ADHD population and by number of biochemical analyses schizophrenia. Similarly, maternal smoking is associated with increased incidence of ADHD. Towards this goal, we propose two Specific Aims in an R21 proposal that we believe may have high impact in the field by developing this model of GxE that can be evaluated mechanistically by targeting the genetic lesion to different neural systems and by receptor signaling assays that are contribute to synaptic plasticity in the striatum. Our strategy is to develop an innovative, integrated approach that capitalizes on the molecular genetic, neuropharmacological and electrophysiological expertise of our laboratories. The Specific Aims are: 1) to evaluate mechanism of GxE effects on synaptic plasticity (long-term depression, LTD) through dopamine D2 receptor signaling and expression and 2) to identify those components of striatal circuitry responsible for deficits in synaptic plasticity by targeting haploinsufficiency of the Snap25 gene to specific inputs to the striatum. Ultimately, we expect that this strategy could be adopted and expanded to define the significant contributions of GxE factors to alterations in gene expression and epigenetic regulation, and the deficits in synaptic plasticity and cognitive behaviors that are the cornerstones of mental illness. Furthermore we believe that this research will aid in the development of better, more targeted therapeutics tuned at a systems level to the neurophysiological basis of neuropsychiatric disorders. PUBLIC HEALTH RELEVANCE: Many prominent neuropsychiatric disorders, such as attention-deficit hyperactivity disorder (ADHD) and schizophrenia, show a clear pattern of inheritance, as well as a striking role for environmental risk factors. Despite this clear evidence for the role of genes x environment interactions, the lack of well-defined animal models has limited our ability to understand the fundamental basis of how these factors interact to promote different clinical presentations of neuropsychiatric disease. Our investigation of a mouse model based on SNAP25 as susceptibility gene and prenatal nicotine exposure would not only help resolve the neurophysiological mechanisms involved, but importantly should aid in the development of better targeted therapeutics based on the brain circuitry revealed by these studies.