Vladislav M. Panin - US grants

[unreadable] DESCRIPTION (provided by applicant): The long-term goal of our research is to understand the role that plays glycosylation in regulating cell interactions during animal development. Mammalian sialylation has become the focus of intensive investigation because of its involvement in important biological processes, such as pathogen-host interactions and the functioning of the immune and nervous systems. Defects in the sialylation pathway have been implicated in multiple pathologies, including tumor metastases, impaired synaptic plasticity, and neuromuscular disorders. At the same time, the complexity of sialylation, and the limit on genetic approaches impose significant difficulties on elucidating biological functions of sialylation in mammals. This project is directed towards a comprehensive understanding of molecular and genetic mechanisms of sialylation in the Drosophila model system. The advantages of this system are based on its advanced genetic approaches, abundance of information on well-documented developmental events, the complete genome sequence, and relatively low genetic redundancy. The proposed multidisciplinary research is aimed at a comprehensive characterization of the Drosophila sialyltransferase gene at the molecular and genetic levels, as well as elucidating the role of sialylation in Drosophila development. One of the specific aims of this project is to comprehensively characterize the sialyltransferase biochemical activity. To this end, the sialyltransferase protein will be expressed in cell culture, purified using affinity chromatography, and assayed for its enzymatic activity. Another specific aim is to investigate in detail the expression pattern of the sialyltransferase (both gene and protein) during different developmental stages. To precisely map the expression of the sialyltransferase, different molecular markers will be used in immunostaining and in situ hybridization analyses. The function of the sialyltransferase will be analyzed at molecular, cellular and organismal levels by comprehensive characterization of the sialyltransferase gene-associated phenotypes obtained by several genetic techniques, including gene targeting and RNAi approaches. Finally, in the framework of this project, the in vivo molecular targets of sialylation will be identified using a proteomics- based approach. This proposed research will elucidate the molecular mechanism and biological role of sialylation in Drosophila and should shed light on biological functions of sialylation in mammals, including humans. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the molecular mechanism(s) controlling stem cell proliferation in response to developmental cues. Stem cells are unique precursors that are self-renewing and give rise to various cell types. Insufficient stem cell division leads to birth defects, while uncontrolled division leads to many types of cancer. Thus, stem ceils have an enormous impact on the health, viability and maintenance of all organisms. In Drosophila, subsets of neuronal stem cells, or neuroblasts, are quiescent upon larval hatching and resume cell division at specific developmental stages; the timing of proliferation is affected by mutations in trol. Trol activates division in quiescent neuroblasts. We have shown that trol encodes a homolog of mammalian Perlecan, a co-receptor for growth factors. Our genetic and biochemical analyses suggest that the signaling pathways activated by two growth factors, Bnl (the Drosophila FGF) and Hh, drive initiation of division in the regulated cells. Signaling by these pathways is modulated by Trol. We have begun to produce fusion proteins and generate domain-specific antibodies. Both types of reagents will be used to probe the structure of mutant Trol proteins and the specificity of Trol-FGF and Trol-Hh binding. We will use the antibodies and a trol-GAL4 transgene to follow trol expression. We will examine how each pathway acts in concert or independently to affect division of specific neuroblasts or neuroblast subsets by altering the levels of ligands and other components, characterizing the expression pattern of pathway proteins, and blocking activation of the FGF pathway. To elucidate how signaling specificity is attained, we will assay for allele-specific interactions between trol and bnl or hh, ascertain the effect of over-expressing trol domains in weak ligand mutants, study the consequences of simultaneously changing levels of both Bnl and Hh, and determine the role of sugar moieties in signaling specificity. These studies will provide important insights into the mechanism(s) by which stem cell division is controlled by developmental cues--a process of fundamental importance in all animals, including humans.

DESCRIPTION (provided by applicant): Our research focuses on the molecular, cellular, and systemic mechanisms underlying the neural functions of glycoprotein sialylation. Although the brain is the organ with the most prominent sialylation in human body, and recent studies implicated sialylation defects in several neurological diseases, the functions of this important type of glycosylation in the nervous system are still poorly understood. The intricacies of glycosylation, increased pleiotropy and redundancy, and limitations of available genetic approaches significantly hinder the research on sialylation in the overwhelmingly complex vertebrate nervous system. Thus, a suitable model system would be an important tool for more efficient and accelerated studies in this area. Here we propose to use Drosophila as a model organism to investigate the neural functions of N-linked sialylation. We previously characterized Drosophila sialyltransferase, DSiaT, a sole sialyltransferase in Drosophila. This enzyme is highly homologous to its human counterpart which also shares with DSiaT several functional properties, including similar acceptor specificity and an elevated expression in the brain. Our recent experiments revealed that the function of sialylation in Drosophila is limited to the nervous system. We found that sialylation regulates neural transmission and the development of neuromuscular junctions. Abnormal sialylation results in Drosophila in prominent neurological phenotypes, including temperature-sensitive paralysis, defects in locomotion, and a significantly shortened life span. Our experiments indicated that a simple N-linked glycoprotein sialylation plays a prominent role in modulating neural activity, which establishes a new paradigm of the involvement of glycosylation in the nervous system regulation. This novel, nervous system-specific function of N-linked sialylated glycans is potentially conserved between flies and humans. The current project will extend our previous research and will investigate (i) the cellular mechanisms underlying the neural function of sialylation in Drosophila, (ii) the molecular mechanisms of sialylation-mediated control of neural excitability, and (iii) the role of sialylation in neural plasticity. We will use a multidisciplinary strategy, combining the advantages of Drosophila model system, including its exceptional amenability to genetic manipulations, exhaustively characterized neural development, low redundancy and pleiotropy of sialylation genes, with well-established electrophysiological and behavioral approaches, cell culture and biochemical techniques, as well as novel technologies for glycan analyses. This project will shed light on the crucial evolutionarily conserved principles of neural regulation and development, which could be useful for biomedical research and relevant therapeutic strategies. Our research will also establish Drosophila as a versatile model system for future studies of the role of glycosylation in the nervous system. PUBLIC HEALTH RELEVANCE: Glycosylation modifies protein functions and profoundly affects the development and physiology of human brain. We will use Drosophila (fruit flies) as an experimentally amenable, genetically tractable and well-studied model organism to elucidate the important functions of glycosylation in the nervous system. Our research will shed light on biological mechanisms that may suggest novel therapeutic strategies for curing neurological diseases with abnormal neural excitability, including as epilepsy and chronic pain.

The main objective of this project is to elucidate functional mechanisms underlying regulation of the nervous system by protein O-mannosylation (POM). POM is an essential type of O-glycosylation that has a profound effect on the development and physiology in a broad range of animals, from Drosophila to humans. Although the spectrum of biological functions affected by POM is wide, so far the only well-studied target of POM is Dystroglycan (Dg). Defects in POM modifications of Dg result in severe muscular dystrophies called dystroglycanopathies. Pathomechanisms associated with POM defects are complex and remain poorly understood, particularly in the nervous system. Recent studies suggested that POM modification affects functions of many proteins, which contributes to pathogenic mechanisms of dystroglycanopathies. However, functions of POM on proteins besides Dg are largely unknown. The complexity of glycosylation and limitations of in vivo approaches create significant challenges for studying POM in mammalian organisms. Here we propose a multidisciplinary project that uses advantages of Drosophila model, including powerful arsenal of genetic approaches, simplified glycosylation and experimental amenability of POM and Dg mutants, to elucidate molecular and cellular mechanisms of Dg- dependent and Dg-independent functions of POM, with the focus on the nervous system and neuromuscular development and physiology. Our preliminary studies suggested that Receptor Protein Tyrosine Phosphatases (RPTPs) are functionally important POM targets and revealed that POM regulates coordinated muscle contractions by affecting communication between sensory neurons and the CNS. We will capitalize on these results while focusing on three specific aims: (1) To analyze the role of POM in regulation of sensory neurons and coordinated muscle contractions. Using live imaging techniques combined with genetic and neurobiological approaches, we will comprehensively investigate the role of POM in communication between sensory neurons, CNS cells and muscles. (2) To investigate the effect of POM on RPTP function. Using in vivo and in vitro approaches, we will investigate how POM affects functions RPTPs at molecular, cellular, and organismal levels. (3) To reveal new molecular targets of POM and elucidate their function in the nervous system. We will use glycoproteomic approaches to identify proteins with POM modifications. We will analyze functions of POM on novel targets in vivo, focusing on proteins that function in the nervous system. We anticipate that this project will establish new paradigms of POM-mediated regulation of the nervous system and will elucidate new evolutionarily conserved, Dg- dependent and independent mechanisms of POM functions, which will shed light on pathomechanisms of human diseases associated with POM abnormalities.