Jorgen Johansen - US grants

A central problem in understanding the development of the brain is to gain insight into the molecular basis for cellular recognition and for how precise neuronal connections are established. During early development axons must pioneer novel pathways through uncharted embryonic landscapes both within the CNS and to and from the periphery. These pathways in turn may serve as a guide for later differentiating neurons, the axons of which have been shown to make highly specific pathway choices and to show selective fasciculation. This process may play a crucial role in the correct wiring of the nervous system. Most hypotheses about the molecular mechanism for selective fasciculation involve specific adhesion or recognition events between axons and/or growth cones mediated by surface macromolecules. However, the challenge has been to test this hypothesis and to identify and characterize such molecules, since the more specific and restricted their expression and distribution is, the lower their abundance. Consequently, a very limited number of molecules has been isolated with proposed adhesion and recognition functions and only a handful of these are confined to subsets of axons and not just involved in general neural cell adhesion. The object of the present proposal is to increase our knowledge of such molecules by determining the molecular structure and function of an antigen recognized by the monoclonal antibodies lan 3-2 and lan 4-2 as well as other antigens which define small subsets of axons forming specific fascicles in the leech. From the amino acid sequence of the antigen, which is a membrane surface glycoprotein, we will analyze the functional implications of its structure. Specifically, we want to test the hypothesis that these antigens are mediating the selective axon fasciculation and thus may represent molecules involved in neuronal recognition and axon guidance. We will also carry out a molecular characterization of the gene locus by exploring its fine structure, its expression and mode of action during development. The promise of cloning these antigens in the leech is that they are specific for a very small and well defined populations of axons and therefore are not likely to be just mediating general adhesion. Our long range goal in analyzing the lan 3-2/4-2 antigen and other leech antigens specific for axons and axonal subsets is to gain basic insights into the functional significance of such molecules, their possible hierarchial organization, functional determinants, and developmental regulation of expression. Since it has been established that many important structural protein sequence motifs have been functionally conserved throughout evolution these investigations should enhance our basic understanding of neuronal recognition and selective fasciculation and provide insights into the underlying causes of aberrant neural connections and abnormal brain development.

A central problem in understanding the development of the brain is to gain insight into the molecular basis for cellular recognition and for how precise neuronal connections are established. During early development axons must pioneer novel pathways through uncharted embryonic landscapes both within the CNS and to and from the periphery. These pathways in turn may serve as a guide for later differentiating neurons, the axons of which have been shown to make highly specific pathway choices and to show selective fasciculation. This process may play a crucial role in the correct wiring of the nervous system. Most hypotheses about the molecular mechanism for selective fasciculation involve specific adhesion or recognition events between axons and/or growth cones mediated by surface macromolecules. However, the challenge has been to test this hypothesis and to identify and characterize such molecules, since the more specific and restricted their expression and distribution is, the lower their abundance. Consequently, a very limited number of molecules has been isolated with proposed adhesion and recognition functions and only a handful of these are confined to subsets of axons and not just involved in general neural cell adhesion. The object of the present proposal is to increase our knowledge of such molecules by determining the molecular structure and function of an antigen recognized by the monoclonal antibodies lan 3-2 and lan 4-2 as well as other antigens which define small subsets of axons forming specific fascicles in the leech. From the amino acid sequence of the antigen, which is a membrane surface glycoprotein, we will analyze the functional implications of its structure. Specifically, we want to test the hypothesis that these antigens are mediating the selective axon fasciculation and thus may represent molecules involved in neuronal recognition and axon guidance. We will also carry out a molecular characterization of the gene locus by exploring its fine structure, its expression and mode of action during development. The promise of cloning these antigens in the leech is that they are specific for a very small and well defined populations of axons and therefore are not likely to be just mediating general adhesion. Our long range goal in analyzing the lan 3-2/4-2 antigen and other leech antigens specific for axons and axonal subsets is to gain basic insights into the functional significance of such molecules, their possible hierarchial organization, functional determinants, and developmental regulation of expression. Since it has been established that many important structural protein sequence motifs have been functionally conserved throughout evolution these investigations should enhance our basic understanding of neuronal recognition and selective fasciculation and provide insights into the underlying causes of aberrant neural connections and abnormal brain development.

DESCRIPTION (from applicant's abstract) The formation of correct neuronal pathways during the early stages of embryogenesis involves a number of navigational strategies. Prominent among them is the extension of growth cones along particular axon tracts, the selection of which is likely to me mediated by a hierarchy of molecular guidance cues. This process may play a crucial role in the correct wiring of the nervous system. The object of the present proposal is to increase our knowledge of such molecules by identifying and dissecting the molecular guidance mechanisms of a well defined population of peripheral sensory neurons in leech which make highly specific pathway choices. The promise of doing this in this system is that several novel molecules - the interactions between which are likely to be involved in this process - have already been identified and that these molecules have some of the most restricted axonal tract distributions yet described. Thus, we will perform a molecular and functional analysis of such proteins which are expressed by small subsets of axons forming specific fascicles in the leech with special emphasis on the cloning of lan3-2/4-2 antigens. In addition, we will molecularly characterize 1) a newly discovered protein with partial thrombospondin homology, L(tsp), which may interact with the lan3-2 antigen, and 2) a 200 kD protein, L(p200) which interacts with calsensin, a small EF-hand calcium-binding protein expressed in a subset of peripheral neurons fasciculating in a single axon tract. In these experiments we will in particular test the hypothesis that the lan3-2 antigen may be functioning both as a growth promoting homophilic adhesion molecule and that it may participate in heterophilic interactions with L(tsp), which is expressed by CNS effects, in a way that helps guide that axons of peripheral sensory neurons to the CNS. We will do this by in vivo function blocking assays with antibody and recombinant protein fragments and by CNS ablation and translocation experiments in the developing embryo. From the proposed experiments we will gain a basic understanding of the structure and functional significance of these molecules, their possible hierarchial organization, and their developmental regulation of expression. As our previous results demonstrate, the relatively simple and accessible system of the leech embryo provides a unique opportunity for identifying molecules mediating axonal pathway formation and to correlate their structure with their function in vivo. Since it has been established that many important structural protein sequence motifs have been functionally conserved throughout evolution, these investigations should enhance our basic understanding of neuronal recognition and selective fasciculation and provide new insights into the underlying causes of aberrant neural connections and abnormal brain development.

DESCRIPTION (From applicant's abstract): The long range objective of this project is to elucidate the functional role of neural molecules in the development of common nerve pathways and selective axon fasciculation. Towards this end by immunoaffinity purification with the mAb Lan3-2, the investigator and associates have identified a novel member of the L1 family of CAMs, Tractin, which is a multiple domain cleaved protein with several unique features. It contains 6 Ig-domains, 4 FNIII-like domains, an acidic domain, 12 repeats of a novel collagen-like proline- and glycine-rich sequence motif, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ-domain binding motif. Tractin is expressed by all neurons but is differentially glycosylated with the Lan3-2 and Laz2-369 glycoepitopes only in sets and subsets of peripheral sensory neurons that form specific fascicles in the CNS. In vivo and in vitro antibody perturbation of these glycoepitopes have demonstrated that they can selectively regulate axonal outgrowth and synapse formation. In addition, at least three other mAbs (Lan2-3. Laz6-212, and Laz7-79) which recognize different glycoepitopes specific to distinct subsets of these neurons have been identified. We will test the hypothesis that these glycoepitopes represent additional posttranslational modifications to Tractin and that such differential glycosylation of a widely expressed neural CAM can functionally assist in regulation neuronal outgrowth and synapse formation of distinct neuronal subpopulations. As Tractin is also expressed by all central neurons these findings suggest that Tractin may function as a major regulator of axon fasciculation, neurite extension, and axonal guidance during early nervous system development. The proposed experiments will test this hypothesis and determine the relative contributions of the different domains of Tractin to these processes in vivo and will identify other proteins with which Tractin interacts to mediate these functions. In addition, expression studies in the S2 cell line will provide novel information about the biosynthesis and mechanisms of posttranslational processing of the L1 family CAMs. Mutations in human and murine L1 lead to severe brain abnormalities; however, the causative developmental mechanisms of these brain defects are not well understood and are likely to involve interaction of L1 with extracellular ligands as well as with intracellular signaling pathways linked to cytoskeletal elements. It is therefore of importance to explore the molecular basis for such interactions in various model systems where such interactions are tractable in order to define the range of structural diversity, functions, and signaling capabilities of L1 family CAMs. Thus, these studies will provide valuable new insights into the underlying causes of aberrant neural connections and abnormal brain development.

The long term objective of this study is to determine how nuclear morphology is regulated during the cell cycle and what role nuclear components may play in establishment of the mitotic spindle. Towards this end, the investigators have identified a novel nuclear protein in Drosophila, Skeletor, which reorganizes from chromosomally associated structure at interphase to a spindle-like structure at metaphase. Double and triple labelings of Skeletor, tubulin, and DNA indicate that the Skeletor spindle may precede the establishment of the microtubule spindle. When this structure is perturbed by anti-Skeletor antibody injection, the investigators observe deterioration of nuclear morphology and a significant decrease in nuclear division. Thus, the investigators hypothesize that there exists a nuclear structure that reorganizes during the cell cycle which plays an essential role in mitotic spindle assembly and/or function yet is distinct from the microtubule spindle. This hypothesis is strengthened by the observation that embryonic preparations treated briefly with nocodazole and which are void of microtubules still show an intact albeit somewhat deformed Skeletor spindle.
The investigators plan to test this hypothesis by performing a number of experiments designed to answer the following key questions: 1) Do anti-Skeletor antibodies identify a previously undescribed nuclear structure? 2) How does this structure reorganize during the cell cycle and does it provide a guide for establishment of the mitotic spindle? 3) Is Skeletor an integral or associated component of this nuclear structure? 4) Does Skeletor function in signaling or effecting the nuclear reorganization events during the cell cycle? 5) What is the effect of perturbing this structure genetically or by other means? 6) What other components interact with or regulate this structure? The information obtained from these experiments will provide major new insights into nuclear architectural remodeling during the cell cycle and microtubule spindle assembly and function during mitosis.

DESCRIPTION (provided by applicant): Project Summary/Abstract A number of molecules with critical roles in chromatin organization, remodeling, and epigenetic modification have been identified in recent years. However, our understanding of which ones are the key regulators or how chromatin structure marked by a specific epigenetic modification is established is far from complete. We have presented evidence that epigenetic histone H3S10 phosphorylation by the JIL-1 kinase at interphase is a key regulator of euchromatic regions by antagonizing heterochromatization and gene silencing in Drosophila. Consequently, to understand regulation of heterochromatin formation and gene silencing in Drosophila, a premier model system for such studies, it will be crucial to determine the molecular mechanisms of the H3S10ph mark's role in this process. Based on our previous findings we propose a model where JIL-1 kinase activity and phosphorylation of histone H3S10 functions to antagonize heterochromatization by regulating a dynamic balance between factors promoting repression and activation of gene expression. The molecular mechanisms underlying this hypothesis will be explored in three specific aims: 1) In the first aim we will test the hypothesis that H3S10 phosphorylation functions to regulate the epigenetic state of euchromatin by using a LacI-tethering system to ectopically induce H3S10 phosphorylation and determine the changes in the distribution of chromatin markers that are diagnostic for active (euchromatic) or silenced (heterochromatic) chromatin. In order to test the inter-relationship of these epigenetic marks, we will furthermore determine whether combinations of targeted histone modifications can counteract or enhance changes in chromatin structure caused by the single modification. 2) In the second aim we will test the hypothesis that epigenetic H3S10 phosphorylation is sufficient to counteract heterochomatic spreading and silencing independently of gross alterations in polytene chromosome morphology. We will use PEV suppression/enhancement as a "read out" for the relative influence of H3S10 phosphorylation on gene expression. To separate out structural and catalytic contributions of JIL-1 we will express truncated and "kinase-dead" JIL-1 proteins transgenically in both wild-type and JIL-1 null mutant backgrounds and quantify the effect on PEV of different reporters. 3) In the third aim we will address the question of whether H3S10 phosphorylation is targeted to specific genomic locations. We will answer this question by specifically mapping interphase JIL-1 and H3S10 phosphorylation sites by ChIP-seq of non-dividing salivary gland chromosomes. We will use microarray analysis to identify genes whose expression levels are regulated by JIL-1-mediated H3S10 phosphorylation. We expect that the proposed studies of H3S10 phosphorylation by JIL-1 will greatly extend our knowledge of how a specific epigenetic mark modulates chromatin structure and gene regulation, a topic that is directly relevant to human development and disease including cancer. PUBLIC HEALTH RELEVANCE: Project Narrative The proposed experiments will greatly enhance our understanding of the molecular mechanisms controlling heterochromatin formation and epigenetic gene regulation by the H3S10 phosphorylation mark. Gene silencing is a critical developmental process relevant to many human health problems that include cancer.