Linda C. Hsieh-Wilson - US grants

[unreadable] DESCRIPTION (provided by applicant): This project seeks to understand the molecular mechanisms by which fucosyl saccharides influence recognition and communication between nerve cells in the brain. Several lines of evidence suggest that fucose alpha (1-2) galactose (Fuc alpha(1-2)Gal) saccharides play an important role in modulating neuronal connections important for long-term memory. Relatively little is known, however, about their precise structures or function in the brain. The proposed research combines chemistry and neurobiology to identify the molecular components - i.e., fucosyl saccharides, glycoproteins and lectins - and to study the mechanisms by which they regulate the flow of information across the synapse. The specific aims of this project are to: 1. Identify Fuc alpha(1-2)Gal lectins in the brain 2. Identify Fuc alpha(1-2)Gal glycoproteins in the brain 3. Investigate whether the Fuc alpha(1-2)Gal functions as a "recognition element" by examining pair wise interactions between identified lectins and glycoproteins and by studying their contribution to neuronal morphology and synaptic transmission 4. Investigate whether the Fuc alpha(1-2)Gal epitope functions as a "targeting element" to regulate the trafficking of glycoproteins to the synapse The long-term goal of this program is to provide a better understanding of the molecular and cellular basis of nerve cell communications and, ultimately, long-term memory formation. Because the program takes a distinctly chemical approach, these studies may ultimately reveal novel points of therapeutic intervention and enable the design of molecules capable of modulating cognition and improving deficits associated with age and neurodegenerative disease. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Developing an understanding of the molecular mechanisms that underlie neuronal communication, and hence form the basis of learning and memory, stands as one of the central challenges of modern science. In this proposal, the focus is on two carbohydrate modifications that play a central role in this process, fucosylation and O-GlcNAc (O-linked N-acetyglucosamine) glycosylation. Protein fucosylation is enriched at neuronal synapses and has been implicated in long-term memory consolidation. O-GlcNAc glycosylation is a dynamic, intracellular modification found on neuronal proteins involved in gene expression, cell signaling, and synaptic plasticity. A major goal of our work is to develop an understanding of the molecular mechanisms by which these sugars influence neuronal communication and information storage. What proteins are modified in the brain, and how do specific carbohydrates regulate the structure and function of neuronal proteins? Are there common themes in the way Nature uses carbohydrate structures to encode functional information? Addressing these questions will be critical for understanding the structure-activity relationships of carbohydrates and their roles in complex brain processes. As our program takes a chemical approach, this work may reveal novel proteins and pathways for therapeutic intervention and aid in the development of new Pharmaceuticals designed to improve cognition deficits associated with aging and neurodegenerative disease. Unlike nucleic acids and proteins, carbohydrate structures are not template-encoded and thus are challenging to detect and manipulate in vivo. As such, new tools are needed to complement the traditional approaches of biochemistry and genetics to advance our understanding of carbohydrates. This work focuses on the development of chemical tools to accelerate the discovery and study of fucosyl and OGlcNAc sugars and their associated proteins. When combined with the power of biochemistry, genetics and neurobiology, these tools will provide new insights into the physiological roles of carbohydrates in the nervous system and uncover novel mechanisms of neuronal communication, learning and memory formation. The major goal of this work is to understand how carbohydrates contribute to the molecular mechanisms that underlie neuronal communication and hence form the basis of learning and memory. Ultimately, our studies should reveal novel targets for therapeutic intervention and may aid in developing new Pharmaceuticals for improving cognition deficits associated with aging and neurodegenerative disease.

DESCRIPTION (provided by applicant): This project will focus on chondroitin sulfate (CS) glycosaminoglycans, a class of polysaccharides that play important roles in development, viral invasion, cancer, and spinal cord injury. CS polysaccharides display diverse sulfation patterns that are spatiotemporally regulated in vivo. However, efforts to identify functions for specific sulfation motifs have been hampered by the structural complexity of CS and a lack of tools. For instance, well-defined CS molecules cannot be purified from natural sources, and as a consequence, the field has been limited to working with heterogeneously sulfated mixtures of compounds. Genetic deletion of one of the sulfotransferase genes responsible for CS biosynthesis can propagate global changes throughout the carbohydrate chain, making it difficult to pinpoint the role of specific sulfation motifs. In this grant, we will combine the tools of organic chemistry and biology to overcome these challenges. We will exploit synthetic chemistry to assemble defined CS structures (both natural and non-natural) to obtain fundamentally new information about the structure- function relationships of CS. When combined with the power of biochemistry, genetics, cell biology, and neurobiology, these molecules will provide new insights into the physiological roles of carbohydrates in the nervous system and uncover novel mechanisms of neuronal growth and repair. As our project takes a distinctly chemical approach, this work may reveal novel proteins and pathways for therapeutic intervention and aid in the development of new pharmaceuticals to stimulate regeneration after injury, aging or disease. PUBLIC HEALTH RELEVANCE: This research seeks to understand how the structure of chondroitin sulfate glycosaminoglycans regulates fundamental biological processes, such as protein recognition and regulation, cell-cell communication, development, and regeneration after injury. Through the discovery of novel small molecules, proteins and pathways involved in these processes, this work may aid ultimately in the development of new therapeutic approaches to a broad range of disease states, such as cancer, infectious and neurodegenerative diseases.

Project Summary/Abstract Carbohydrates comprise one of the largest, most diverse collections of biologically active molecules. However, relative to other biomolecules such as nucleic acids and proteins, carbohydrates remain poorly understood due to challenges in their detection, synthesis, and analysis. The broad objective of this program is to develop chemical approaches to advance a fundamental understanding of the roles of carbohydrates in biology and disease. In the last granting period, we developed a novel Networking of OGT Interactors and Substrates (NOIS) method to study the biological functions of O-linked ?-N-acetylglucosamine (O-GlcNAc) glycosylation. O-GlcNAc is an abundant, essential post-translational modification that is emerging as a key regulator of many physiological functions, ranging from epigenetic and transcriptional gene regulation to insulin signaling, cancer cell metabolism, and neurodegeneration. Our NOIS approach combines new chemoproteomic tools, genetic engineering, MS analysis, and bioinformatics methods to examine interconnections between the interactors and substrates of O-GlcNAc transferase (OGT) across the proteome both in vitro and in vivo. These analyses have revealed novel, unexpected functions for the O-GlcNAc modification and highlighted potential mechanisms to explain the unique specificity of OGT. In the coming granting period, we will expand this approach to important physiological and disease contexts and tackle the next set of critical barriers in the field. In Aim 1, we will develop next-generation chemoproteomic tools and MS methodologies to quantify changes in O-GlcNAc networks in response to biological stimuli. In Aim 2, we will apply our quantitative NOIS method to study the dynamics of O-GlcNAc networks during both normal and excitotoxic neuronal stimulation, and the network perturbations in an in vivo model of late-onset Alzheimer's disease (AD) and metabolic syndrome/T2DM-associated dementia. In Aim 3, we will demonstrate how NOIS can produce novel, tractable hypotheses and test those hypotheses to establish previously undiscovered functions for O-GlcNAc in synaptic plasticity. We will also use NOIS to explore ways to selectively modulate OGT's activity toward specific substrates. Finally, in Aim 4, we will apply similar proteomic and bioinformatics approaches to investigate the crosstalk between O-GlcNAc and phosphorylation, as well as its impact on neuronal signaling pathways critical for neuronal communication, homeostasis, and synaptic plasticity. Together, the proposed studies will provide a unified approach to track dynamic O-GlcNAcylation events across the proteome and identify physiologically important and/or disease-causing O-GlcNAcylation events. In turn, this information should provide new potential therapeutic targets or approaches to combat progressive neurodegeneration, Alzheimer's disease, and related dementias.

? DESCRIPTION (provided by applicant): This project will focus on chondroitin sulfate glycosaminoglycans (CS GAGs), a class of polysaccharides that play important roles in development, viral invasion, cancer, and spinal cord injury. CS GAGs display diverse sulfation patterns that are spatiotemporally regulated in vivo. However, efforts to identify functions for specific sulfation motifs have been hampered by the structural complexity of CS and a lack of tools. In this grant, we will combine the power of both organic chemistry and biology to overcome these challenges and identify novel functions for specific motifs in the nervous system. The broad objectives of this program are to: (1) advance a fundamental understanding of the structure-function relationships of CS GAGs, (2) understand the roles of CS GAGs in neuroplasticity and regeneration, and (3) develop new approaches to study and manipulate GAG-mediated biological processes, with the long-term goal of stimulating synaptic plasticity and neuronal repair. In the last granting period, we developed a set of chemical tools to study specific sulfation motifs and discovered that a particular motif, CS-E, inhibits axon regeneration after spinal cord injury. Blocking the CS-E motif using an anti-CS-E antibody stimulated axon regeneration in vivo. Moreover, we found that this same motif repels axons and plays a critical role in neural circuit formation during brain development. An important observation from this work was that the activity of CS-E required its interaction with cell-surface receptors and activation of specific inhibitory signaling pathways in neurons. In the present grant, we will develop new approaches to modulate the interactions of CS-E with neuronal receptors, including the viral-mediated delivery of single-chain anti-CS-E antibodies, small- molecule sulfotransferase inhibitors, and glycopolymer mimetics (Aim 1). We will study how CS-E regulates protein signaling complexes, with a particular focus on semaphorin-3A/neuropilin-1/plexin A (Sema3A/Nrp1/PlxnA) and ephrin/Eph receptor (Efn/Eph) complexes (Aims 2a and 3a). Finally, we will investigate the ability of the agents developed in Aim 1 to promote neuroplasticity in the visual cortex (Aims 2b,c) and axon regeneration after spinal cord injury (Aim 3b). These studies are expected to provide new chemical tools to advance an understanding of GAGs and fundamentally change how CS GAGs are viewed - from being static, passive molecules to ligands that actively regulate important signaling pathways. Finally, if successful, the agents developed in Aim 1 could lead to novel therapeutic strategies for stimulating neuronal plasticity and repair in the case of aging, injury, and disease.

? DESCRIPTION (provided by applicant): Heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans play important roles in many physiological and pathological events, such as cell division, inflammation, neuronal development, and cancer metastasis. Naturally existing HS and CS display a diverse range of sulfation patterns. While this structural diversity bestows HS and CS with the ability to interact with many proteins, it greatly hinders the ability to decipher their structure-function relationships. In order to dramatically advance an understanding of the biological functions of glycosaminoglycans, it is critical to access large, structurally diverse libraries of HS and CS oligosaccharides bearing well-defined sulfation sequences. To date, synthetic methodologies toward HS and CS are mostly target oriented, resulting in only small sets of oligosaccharides. Furthermore, it remains difficult to prepare HS and CS sequences longer than a dodecasaccharide. To address these challenges, three research groups with strong, complementary expertise in HS and CS synthesis and biology have joined forces to accomplish the following aims. In Aim 1, new synthetic strategies are proposed to accelerate the synthesis of HS oligosaccharides. Methodologies will be developed to prepare the first comprehensive library of 256 HS tetrasaccharides representing all of the possible 2-O, 6-O and N sulfation motifs, along with a library of structurally diverse 3-O sulfated tetrasaccharides and HS hexasaccharides. In Aim 2, we propose new efficient, cost-effective routes to access the first comprehensive library of CS tetrasaccharides bearing all of the possible mammalian sulfation sequences. In Aim 3, HS/CS oligosaccharide-based polymers and head-to-tail multimers will be prepared to enable access to structures containing homogeneously sulfated glycans with sizes approaching natural polysaccharides. These mimetics will possess similar domain structures and multivalent properties found in naturally existing CS and HS polysaccharides. In Aim 4, we will validate the hypothesis that our molecules can recapitulate the biological functions of HS and CS polysaccharides using well-established assays, including anticoagulation, neuronal growth, and protein-binding assays. Furthermore, we will explore the potential for these molecules to selectively target a clinically important family of proteins, the fibroblast growth factors. Together, this project will provide faster, more affordable syntheses of HS and CS, greatly expand the chemical space currently accessible by synthesis, enable the first direct, in-depth comparisons between HS and CS, and provide novel agents to control the activities of these biomedically important molecules.

Project Summary Glycosaminoglycans (GAGs) play important roles in many physiological and pathological events such as cell division, inflammation, neural development, and cancer metastasis. The long polysaccharide chains of GAGs contain various sulfated disaccharides that are organized into sulfate-rich and under-sulfated domains. This rich structural diversity enables GAGs such as heparan sulfate (HS) to interact with numerous proteins and regulate key signaling pathways. However, efforts to understand their structure-function relationships and harness their therapeutic potential have been hampered by the chemical complexity of GAGs and a lack of tools. At present, there are no tools to manipulate the interactions of GAGs with specific proteins of interest, thus complicating efforts to pinpoint their precise roles. The goal of this project is to develop novel chemical probes for selectively modulating GAG activity. We will use a directed evolution-based approach to create a new class of GAG mimetics ? multivalent glycopeptides appended with short, active HS motifs ? to modulate specific HS-protein interactions. Random sampling of peptide sequences by directed evolution should allow for the selection of structures containing the ideal number and arrangement of HS motifs. In addition to optimal HS clustering, selected peptides should contain peptide motifs recognized by the protein of interest, thus generating highly specific GAG probes. In Aim 1, we will work in collaboration with the Krauss laboratory to develop the approach and generate HS mimetics that interact selectively with fibroblast growth factor 2 (FGF2), a key growth factor involved in cell migration, angiogenesis, and differentiation. In Aim 2, we will evolve glycopeptides that bind to specific forms of tau, a microtubule-associated protein linked to dementias such as Alzheimer's disease and Parkinson's disease. We will use these HS mimetics to understand the mechanisms underlying tau uptake into neurons and neurodegeneration. In addition, we will explore whether our mimetics can block the intercellular spreading of tau and its pathogenic consequences. In Aim 3, we will evolve glycopeptides that bind chemokines (specifically CXCL9, CXCL10 and CXCL11), a class of therapeutically important proteins that are key mediators of inflammation. Our probes should provide unique insights into the paradoxical functional redundancy of chemokines, enabling us to tease apart their individual roles. Together, these studies will produce a novel class of GAG-based probes for understanding the physiological functions of GAGs and may ultimately lead to new therapeutic leads or approaches to diseases such as cancer, neurodegenerative diseases, inflammatory and autoimmune disorders, and infectious diseases.