Neuroplasticity and chondroitin sulfate

Chondroitin sulfate glycosaminoglycans (CS GAGs) are a family of linear, sulfated polysaccharides that are covalently linked to core proteins to form CS proteoglycans (CSPGs) on the cell surface. In the central nervous system (CNS), CSPGs make up a large portion of the extracellular matrix and interact with hundreds of structural proteins and signaling molecules to regulate key processes such as cellular maturation and differentiation, neuroinflammation, and neuronal growth during development. As part of our characterization of the structure-function relationships of CS, our lab identified the CS sulfation motif E (CS-E) inhibits neuronal regrowth and recovery. This motif is found abundantly in glial scars that form after CNS injury, and ablation of the CS-E motif results in a restored ability for neurons to regrow axons. These findings highlight the therapeutic potential of studying CS GAGs as potential targets for intervention. Thus, our lab is focused on continuing to develop new tools to facilitate discovery of other functions of CS GAGs and highlight avenues for stimulating neuronal recovery in CNS injury and neurodegenerative disease.

Antibodies specific to CS GAG sulfation motifs
We have raised highly selective monoclonal antibodies against the CS motifs, including CS-A, CS-C, and CS-E, using synthetic CS tetrasaccharides as antigens. We are now generating single-chain variable fragment antibodies (scFv) and establishing viral delivery systems for the blocking of CS motifs in in vivo models of multiple sclerosis, spinal cord injury, and stroke to better understand the mechanisms by which the motifs promote or inhibit neuroregeneration and measure functional recovery.

Brown, J. M. et al., "A Sulfated Carbohydrate Epitope Inhibits Axon Regeneration After Injury," Proc. Natl. Acad. Sci. U. S. A. 2012. DOI: https://doi.org/10.1073/pnas.1121318109

GAG photoaffinity probes for identifying GAG binding proteins
GAG-protein interactions in the extracellular matrix mediate essential biological and pathological processes. However, mapping GAG-protein interaction networks is challenging as they are often context dependent, low affinity, and involve receptors and extracellular matrix-associated proteins. In the Hsieh-Wilson group, we have developed CS-E polysaccharide photo-affinity probes and carried out the first proteome-wide identification of CS-E binding proteins in live cells. We have identified a number of previously uncharacterized interactions between CS-E and extracellular proteins. We are currently characterizing these interactions to understand their roles in the regulation of important biological processes such as neuroplasticity, synapse development, and neuroinflammation.

Joffrin, A. M.; Hsieh-Wilson, L. C., "Photoaffinity Probes for the Identification of Sequence-Specific Glycosaminoglycan-Binding proteins.” JACS 2020. DOI: https://doi.org/10.1021/jacs.0c06046

Understanding the role of CS in perineuronal nets 
Perineuronal nets (PNNs) are extracellular matrix structures that condense around neurons. PNNs regulate synaptic connections and plasticity and modulate behaviors such as learning, memory, and anxiety. CSPGs are a major component of PNNs, and the specific sulfation motif of CS-A has been shown to regulate PNN formation. We are interested in understanding the molecular mechanisms that govern PNNs in the central nervous system. From this understanding, we will provide a framework to manipulate these structures therapeutically and re-open windows of plasticity in the adult, after injury or disease.

Dick, G. et al., "Semaphorin 3A Binds to the Perineuronal Nets via Chondroitin Sulfate Type E (CS-E) Motifs in Rodent Brains," J. Biol. Chem. 2013. DOI: https://doi.org/10.1074/jbc.M111.310029
Huang, H., et al., “Chondroitin 4-O-Sulfation Regulates Hippocampal Perineuronal Nets and Social Memory,” Proc Nat Acad Sci 2023.
DOI: https://doi.org/10.1073/pnas.2301312120.

Small molecule inhibitors
We are developing small molecule tools for interrogating GAG sulfation-dependent processes. Toward this goal, we have recently developed the first cell-permeable, small-molecule inhibitor selective for GAG sulfotransferases, including Chst15, the sulfotransferase responsible for biosynthesis of CS-E. We demonstrated that the molecule specifically inhibits GAG sulfotransferases in vitro, decreases CS-E and overall sulfation levels on cell-surface and secreted chondroitin sulfate proteoglycans (CSPGs), and reverses CSPG-mediated inhibition of axonal growth. This may represent a novel therapeutic approach for neuroregeneration.

Cheung, S. T. et al., "Discovery of a Small-ˇMolecule Modulator of Glycosaminoglycan Sulfation,” ACS Chem Biol. 2017. DOI: https://doi.org/10.1021/acschembio.7b00885

Cell surface remodelling
We are developing novel tools and strategies to remodel the cell surface of various cell-types by introducing structurally defined GAGs. As such, we have developed liposome-GAG conjugates as a facile method to insert specific GAG motifs, such as CS-A, CS-C, or CS-E, onto neuronal cell surfaces, enabling modulation of signaling pathways and downstream control of axonal growth. We plan to leverage this to elucidate the ability of specific CS motifs to direct cellular events.

Pulsipher, A. et al. "Directing Neuronal Signaling through Cell-Surface Glycan Engineering," J. Am. Chem. Soc. 2014. DOI: https://doi.org/10.1021/ja5005174

Design of CS mimetic glycopolymers
We are developing glycopolymers that are more readily synthesized than polysaccharides and have tunable chemical and biological properties. The interactions of these glycosaminoglycan mimetics with proteins are being analyzed to determine the role of specific sulfation patterns and molecular architecture in regulating important biological processes such as axon guidance, growth factor signaling, and inflammation.

​Lee, S-G. et al. "End-functionalized glycopolymers as mimetics of chondroitin sulfate proteoglycans." Chem. Sci. 2010, 1, 322–325 DOI: https://doi.org/10.1039/C0SC00271B
​Rawat, M. et al. "Neuroactive Chondroitin Sulfate Glycomimetics." J. Am. Chem. Soc. 2008, 130, 2959–2961. DOI: https://doi.org/10.1021/ja709993p

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