Metabolic labeling of neuroglycans with unnatural sugars

Abstract

Glycans are intimately involved in several facets of neuronal development and neuropathology. However, the roles of glycans remain elusive compared with those of proteins or lipids because of their diverse and dynamic nature. Method for investigating their functions, using a lectin or eliminating glycosyltransferase gene, had been limited. Metabolic labeling strategy developed in 1997, incorporation of unnatural monosaccharides in the biochemical synthesis of glycans as a chemical reporter, is a powerful tool for exploring the functions of cell-surface glycoconjugates, because it can be chemically modified glycans, directly, without usage of protein or gene.In the neuronal development, the posttranslational modification of neural cell-adhesion molecule (NCAM) with polysialic acid (PSA) play an important role by regulating adhesions of NCAM. A tissue-based strategy, incorporation of a chemical reporter metabolized by hippocampal tissues before dissociation into individual cells, were developed for metabolically incorporating an unnatural monosaccharide, peracetylated N-azidoacetyl-D-mannosamine, in the sialic acid biochemical pathway to present N-azidoacetyl sialic acid to PSA-NCAM. Although significant neurotoxicity was observed in the conventional metabolic labeling that used the dissociated neuron cells, neurotoxicity disappeared in this modified strategy, allowing for investigation of the temporal and spatial distributions of PSA in the primary hippocampal neurons. PSA-NCAM was synthesized and recycled continuously during neuronal development, and the two-color labeling showed that newly synthesized PSA-NCAMs were transported and inserted mainly to the growing neurites and not significantly to the cell body. The spatial distribution of cell‐surface glycoconjugates in the brain changes continuously, reflecting neurophysiology especially in the developing phase, but their functions and fates mostly remain unexplored. Their spatiotemporal distribution is particularly important in polarized neuronal cells, such as cerebral cortical neurons composed of a soma and neurites. The multiplexed information on their spatiotemporal distribution on polarized cortical neurons were obtained by dual-labeling sialic acid (Sia5Ac) and N‐acetylgalactosamine/glucosamine (GalNAc/GlcNAc) using a neurocompatible strategy of metabolic glycan labeling, metabolism‐by‐tissues (MbT). The analyses showed the preferentially distinct distribution of each saccharide set at the late developmental stage after randomized, heterogeneous distribution at the early stage, suggesting that Sia5Ac and GalNAc/GlcNAc are translocated anisotropically during neuronal development.Although the tissue-based metabolic labeling strategy was developed, the metabolic labeling of surface glycans in primary neurons is still a difficult task because of the neurotoxicity of unnatural monosaccharides that are used as a metabolic precursor, hindering the progress of metabolic engineering in neuron-related fields. Therefore, we report a neurosupportive, neuron–astrocyte coculture system that neutralizes the neurotoxic effects of unnatural monosaccharides, allowing for the long-term observation and characterization of glycans in primary neurons in vitro. Polysialic acids in neurons are selectively imaged, via the metabolic labeling of sialoglycans with peracetylated N-azidoacetyl-D-mannosamine (Ac$_4$ManNAz), for up to 21 DIV. Two-color labeling shows that neuronal activities, such as neurite outgrowth and recycling of membrane components, are highly dynamic and change over time during development. In addition, the insertion sites of membrane components are suggested to not be random, but be predominantly localized in developing neurites. A semi-three dimensional, neuron-culture scaffold is designed and fabricated for induction of neuronal-circuit regeneration and advanced investigation of neuro-glycan, where astrocytes are encapsulated in the alginate microfibers. The astrocyte-encapsulating microfibers accelerate the neurite outgrowth, facilitate the synaptic formation, and guide the neurite elongation, without physical contact with neurons. The astrocytes in the microfibers, located nearby neurons in 100 μm, could support neuronal physiological activity and eliminate the toxicity of unnatural monosaccharide, peracetylated N-azidoacetyl-D-mannosamine. Therefore, spatial distributions of surface-glycoconjugates of hippocampal neurons cultured in three dimensional environment were showed. This study suggests a pivotal scaffold for advanced development of cell-based therapeutics for neural injuries, such as spiral cord injury, and a new research platform, advanced 3D systems, for metabolic-labeling studies of glycans in primary neurons.