Targeting Neuronal Degeneration in Gaucher Diseasese
Gaucher Disease (GD) is a lysosomal storage disorder caused by homozygous mutations in the GBA gene, which encodes the enzyme glucosylceramidase (GCase) responsible for the final step in glycosphingolipid degradation. Two forms of GD are neurodegenerative and have been linked to an elevated risk of Parkinson’s Disease (PD). Notably, heterozygous GBA mutations that do not cause GD are also a risk factor for PD. Non-neurodegenerative GD can be treated by enzyme replacement therapy using intravenously delivered recombinant GCase, but the brain is not readily accessible through this route. To target neurodegenerative GD and PD, we plan to use the small molecule Guanidinylated Neomycin (GNeo) to enhance GCase uptake and delivery to the brain. GNeo binds cell surface heparan sulfate and facilitates uptake and lysosomal delivery of molecular cargoes, including proteins. Indeed, previous work using intranasal delivery in mice showed that GNeo-iduronidase conjugates enter the brain to a much greater extent than unmodified enzyme. The plan for the current project is to prepare NHS-modified GNeo for conjugation to exposed lysine residues on recombinant GCase, and then use the GNeo-GCase conjugates to treat human Gaucher fibroblasts and neurons obtained from a mouse model of Gaucher disease. Cell uptake will be confirmed using activity assays performed in cell lysates along with direct visualization in cultured neurons using a fluorophore-modified form of GNeo. At this point, both NHS-GNeo and recombinant GCase have been successfully prepared and the conjugation reaction has been optimized. Conjugates retain full enzymatic activity, and plans to treat cells are imminent.
Julia Callender, PhD | Mentor: Christina Sigurdson
Manipulating PrP glycan structure to understand toxic signaling pathways driving prion-induced neurodegeneration
Prions cause a rapidly progressive neurodegenerative disease characterized by an exponential increase in prion aggregates as well as a spongiform encephalopathy, dystrophic neurites, and neuronal death. These processes depend on the neuronal expression of the cellular glycoprotein, prion protein, PrPC, however, the mechanism through which PrP signaling or aggregation contributes to neurodegeneration remains unclear. Previous work has shown that glycan modifications may impact PrP aggregation and neuronal toxicity. To investigate the role of glycans in prion-induced toxicity, we have engineered a new knockin mouse model introduces an additional glycan on the amino terminus of PrP. This mouse develops spongiform encephalopathy in the absence of PrP aggregates, thus uncoupling PrP aggregation from neurotoxicity. Here we will use this new mouse model to investigate the mechanism through which PrP glycans contribute to prion-mediated neurotoxicity and neurodegeneration.
- Received poster award at Society for Glycobiology conference, November, 2020
- Award received for abstract at UCSD Pathology Department Research Retreat, August 15, 2020
- Award received for abstract at the NIH/FDA Glycoscience research day, May 15, 2020
Dillon Chen, MD | Mentor: Ajit Varki
Characterize the roles of polysialic acids and Sialic acid-binding immunoglobulin-type lectins (SIGLECs) in the brain
Perinatal and neonatal brain injury often leads to long-term morbidity including neurodevelopmental impairment. Understanding the effects of injury during these critical periods of brain development may result in improved morbidity and mortality for infants with brain injury. One of the most fundamental building blocks of the brain is the sialic acid and a major protein-bound sialic acid in the brain is polysialic acid (polySia). In the adult mouse brain, polySia has been shown to be involved in neurogenesis and, with its interacting partner the sialic acid-binding immunoglobulin-like lectin, modulate inflammation. In my studies, I hope to characterize and explore the functions and expression levels of polySia, Siglec-11 and Siglec-16 in the developing brain, especially in the setting of neural injury.
-Received industry sponsored research agreement; Aviceda Therapeutics, Exploring roles of polysialic acid and sialic-acid binding Ig-like lectins (Siglecs) in neurodevelopment, neuroinflammation and response to injury, January 2020
Sun-Mi Choi, MD, PhD | Mentor: Victor Nizet
Upper airway mucosal responses to MRSA in CRS
Chronic Rhinosinusitis (CRS) affects 10% of adults and Staphylococcus aureus (SA) colonization is increased in CRS. One hypothesis to delineate the role of SA in pathogenesis of CRS is that SA stimulates production of c-type lectin Reg3g which binds to peptidoglycan layer of Gram+ bacteria along with type 2 inflammatory cytokines (thymic stromal lymphopoietin [TSLP] and IL-33) that causes hyper production of mucins (Muc5AC and Muc5B) resulting in chronic inflammatory state involving aberrantly activated innate immune and T cells.
- Received scholarship from Western Society of Allergy, Asthma & Immunology Emerging Allergist
Graham Heberlig, PhD | Mentor: Michael Burkart
Structural examination of lipid A biosynthesis complexes
Lipid A, also known as endotoxin, is a glucosamine based saccharolipid essential for the growth of most gram-negative bacteria. It serves as a hydrophobic anchor for lipopolysaccharide (LPS) which is required for virulence. Lipid A is biosynthesized via the Raetz pathway which intersects with fatty acid biosynthesis (FAB) through the incorporation of 3-hydroxy-acyl chains donated by acyl carrier proteins (ACPs). Chemical synthesis of ACP-linked probe molecules will facilitate structural (re)characterization of the four ACP-acyltransferase complexes required to generate mature Lipid A. These structural studies may provide new strategies toward antibiotic and vaccine development.
So-Young Kim, PhD | Mentor: Mark Fuster
Glycosaminoglycan-based lung cancer immunotherapy
Lung cancer is the leading cause of cancer death in the U.S. and worldwide. Dendritic cells (DCs) recognize tumor antigen and instruct T cells to initiate antitumor immunity. In a tumor microenvironment, however, dendritic cells are suppressed from properly expressing mature phenotype to carry on this function. Heparan sulfate (HS) glycosaminoglycan, when altered on the DC surface, restores mature DC phenotype and inhibits immature DC trafficking to the draining lymph nodes demonstrating anti-tumor therapeutic potential, however, its effect on T cell associated lung cancer immunity has not been previously investigated. In my studies, I will uncover how DC specific HS alteration will influence the antitumor associated T cell function and downstream signaling pathways. Additionally, genetic and chemical strategies will be developed targeting DC HS.
Ryan Porell, PhD | Mentor: Kamil Godula
Design and synthesis of heparan sulfate macrophage-targeted glycopolymer mimetics to reprogram tumor microenvironments
Cancer cells demonstrate a unique ability to evade and manipulate immune cells into assisting their proliferation and metastasis through the secretion of growth factors (GFs) and anti-inflammatory cytokines. Heparan sulfate proteoglycans (HSPGs) engage and act as reservoirs for several tumor-proliferative GFs and immune suppressive cytokines. High heparinase expression in tumor microenvironments induces release of these factors to accelerate tumor severity. My research involves synthesizing HS-mimetic nanomaterials, which sequester GFs and cytokines back from cancer cells. These HS-mimetics also contain lysosome targeting glycans (mannose), which transport the HS-mimetics with bound GFs to macrophage lysosomes, thus eliminating them from the tumor microenvironment. Our synthetic mannosylated targeting polymers engage macrophage mannose receptors (CD206), internalize to lysosomes, and slightly activate macrophages based on cell surface phenotype markers. The challenge of attaching full HS chains or HS disaccharides to our mannosylated polymers motivated me to take an alternative approach with a modular design making separate mannosylated-cyclodextrin and HS-adamantane molecules, which engage through host-guest interactions. Analysis of our host-guest designed bifunctional glycopolymers is currently underway. We hypothesize that our multifaceted approach focused on degradation of growth and immune suppressive factors with concomitant immune activation will become a powerful technique towards thwarting cancer proliferation and metastasis.
Ruth Siew, MD | Mentor: Victor Nizet
Sialic acid interactions in cystic fibrosis
Cystic fibrosis is caused by a mutation in the CFTR gene that results in a multi-organ system disease with the primary cause of morbidity from severe, chronic respiratory infections. The opportunistic pathogen,
Pseudomonas aeruginosa, is an early colonizer of the cystic fibrosis lung and causes inflammation and structural lung damage. It produces sialidase as a pathogenic virulence mechanism where desialylation of the airway mucosal surface allows for the colonization of
P. aeruginosa and biofilm formation. There has been limited research in the area of bacterial sialidase as it contributes to the pathogenesis of infection, therefore I am interested in further exploring bacterial sialidase as it relates to the host immune system. CFTR function has also been linked to the sialic acid N-glycolylneuraminic acid (Neu5Gc), which differs from the human-dominant form N-acetylneuraminic acid (Neu5Ac) by one oxygen atom due to lack of the enzyme CMP-N-acetylneuraminic acid hydroxylase (CMAH). I seek to understand how this difference applies to lung epithelial cells with the CFTR mutation. As the Varki lab has had extensive experience with this glycosylation change from Neu5Gc to Neu5Ac, they have also been successful in recapitulating a cholera-like phenotype in a mouse model using a Cmah null mouse. There are currently no mouse models available that have been able to simulate chronic lower respiratory tract infections such as those seen in humans. By crossing the Cmah mouse line with the most common mutation in cystic fibrosis (CFTR delF508), I hope to create a humanized mouse model of disease for cystic fibrosis.
Michelle Ducasa, PhD | Mentor: Philip Gordts
The role of heparan sulfate proteoglycans in non-shivering thermogenesis
The present worldwide obesity epidemic is associated with a sharp increase in its metabolic complications, including a dramatic surge in Type 2 Diabetes. Increasing energy expenditure has emerged as an approach to treat obesity. Most of this energy expenditure takes place in mitochondria, where energy is generated through the electron transport chain, which creates a proton gradient across the inner membrane to drive the synthesis of ATP. This process can be uncoupled, resulting in the consumption of energy and generation of heat. Thermogenic uncoupling of mitochondrial oxidative phosphorylation is largely but not exclusively mediated by the thermogenic protein UCP1 in brown and beige fat. Activating and recruiting brown and beige fat is a major pharmacological focus for obesity and the associated metabolic disorders. Previous studies have shown that heparan sulfate proteoglycans (HSPG) play a role in insulin sensitivity and adipocyte differentiation. Their role in cytokine and growth factor presentation and their abundance in the extracellular environment place HSPGs in a unique position to modulate adipocyte phenotypes. Thus, the goal of this project is to understand if heparan sulfate proteoglycans contribute to adipose tissue plasticity and thermogenic potential.
Richard Sanchez, PhD | Mentor: Gulcin Pekkurnaz
O-GlcNAc dependent cytoskeletal influences on mitochondrial localization under varying cellular metabolic states
Intracellular O-GlcNAc is a single unelongated glycolytic molecule that is added on to proteins as a post-translational modification (PTM) on serine and threonine residues. The enzymes responsible for the addition and removal of O-GlcNAc are O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) respectively. In eukaryotes OGT is a major nutrient sensing enzyme that is expressed in every cell type but is most abundantly expressed in the brain and pancreas. OGT’s catalytic activity is regulated by intracellular concentrations of UDP-O-GlcNAc which is synthesized by varying metabolic substrates through the hexosamine biosynthesis pathway allowing its concentration to fluctuate in response to nutrient availability. Our lab has shown that OGT can regulate mitochondrial localization and metabolic efficiency in neurons. Expanding upon these results we have identified cytoskeletal regulating protein candidates that are susceptible to O-GlcNAcylation using mass spectrometry on isolated neuronal mitochondria. My focus will be on the role of these cytoskeletal proteins in the context of mitochondrial positioning and metabolic compartmentalization under the influence of O-GlcNAcylation. In addition, even though OGT is a single gene it codes for three splice isoforms all of which are capable of O-GlcNAcylating proteins. I will define which OGT isoforms are influencing metabolic efficiency. Overall, these studies will provide a more comprehensive understanding of the metabolic regulatory factors that influence cellular metabolic homeostasis, helping in elucidating human metabolic disorders that range from diabetes to neurodegeneration.
Ryan Weiss, PhD | Mentor: Jeff Esko
CRISPR-Cas9 Dissection of Heparan Sulfate
Heparan sulfate proteoglycans (HSPGs) are expressed on all animal cells and in the extracellular matrix. Each HSPG consists of a core protein with one or more covalently attached linear heparan sulfate (HS) chains composed of alternating glucosamine and uronic acids that are heterogeneously N- and O-sulfated. These complex cell surface carbohydrates regulate important biological processes including cell proliferation and development through their interaction with many matrix proteins and growth factors. The arrangement and orientation of the sulfated sugar residues of HS specify the location of distinct ligand binding sites on the cell surface, and these modifications can vary temporally during development and spatially across tissues. While most of the enzymes involved in HS biosynthesis have been studied extensively, less information exists regarding the mechanisms that give rise to the variable composition and binding properties of HS. The overall goal of this project is to understand the genetic factors that control the formation of heparan sulfate in mammalian cells. A genome-wide CRISPR/Cas9-mediated screen will be developed to uncover novel genes other than those encoding known HS biosynthetic enzymes. A lentiviral single guide RNA (sgRNA) library will be utilized to knock down gene expression across the entire genome in a human cancer cell lines. Subsequently, a high-throughput screen will be adapted to identify sgRNAs that induce resistance to cytotoxins whose action depends on HSPGs or decrease binding of HS-dependent ligands to the cell surface. Overall, these studies will provide a better understanding of the genetic regulatory factors involved in HS biosynthesis, as well as lead us to methods to manipulate HS and its activities in other cellular processes that go awry in human diseases.