Improving Brain Recovery Through Glycoengineering

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Project Summary This project is based on recent advances in metabolic glycoengineering (MGE), a technology platform where non-natural monosaccharides intercept the biosynthetic pathways for cell surface-displayed glycans. As a result, chemical functionalities not naturally found in carbohydrates are installed in the glycalyx, which can alter cell adhesion, receptor activity, and downstream events (e.g., apoptosis, differentiation, and motility). In previous work, we developed the N-acetylmannosamine (ManNAc) analog “Ac5ManNTGc” to install thiol groups into sialic acids in human embryonic cells and found that – when the cells were grown on a “high affinity” surface (e.g., gold, which forms coordinate covalent bonds with thiols) – Wnt signaling was upregulated in the absence of extracellular Wnt proteins and neuronal differentiation was induced. In vivo translation of this approach, however, was hindered by the requirement for a non-degradable gold scaffold. We recently overcame this impediment by designing new ManNAc analogs with thiols presented on longer linkers, which extends this functional group further away from the core monosccharide and increases analog potency. Critically, the new analogs provide pro-neurogenic activity in the absence of a scaffold thereby simplifying in vivo translation. This project will explore analog mechanism in hNSCs in Specific Aim 1; this aim will define how the chemical structure, kinetics, and dose of thiol-modified ManNAc analogs (along with chemically inert size-matched controls) modulate cellular glycans in Aim 1a; evaluate changes to cell adhesion and motility in Aim 1b, and evaluate the differentiation of human neural stem cells (hNSCs) in Aim 1c. Next, in Specific Aim 2, we will apply the optimized analog-treatment conditions to improve neural regeneration in a rat cardiac arrest (CA) model of brain injury by transplanting MGE-modified into injured animals. We will compare hNSCs treated with our new thiol-modified analogs with appropriate controls on functional recovery after CA by evaluating survival, adhesion, distribution, and migration of transplanted hNSCs in rat brain. In Specific Aim 3, we will evaluate biochemical (Wnt signaling and cadherin involvement) and cellular (tissue infiltrating immune cells) level mechanisms we propose contribute to the healing effects of MGE in brain injury recovery (in Aim 3a). Finally, in Aim 3b we will characterize cell-wide “glycosites” by mass spectrometry and use glycobioinformatics analyses to identify unknown biochemical mediators of MGE. Specifically, we anticipate identifying new mediators of the beneficial effects of MGE in the implanted hNSCs as well as trans-acting host proteins. We hypothesize that thio-analogs modulate hNSC fate through a complex combination of receptor- specific effects on cell signaling and adhesion providing a pleiotropic suite of healing effects that cannot be achieved through conventional therapies. Accordingly, our innovative approach opens a new avenue to improve stem cell therapy with our new thiol-based MGE technique.