Small but big leaps towards neuroglycomics: exploring N-glycome in the brain to advance the understanding of brain development and function (2024)

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Glycosylation is a process that involves the addition of sugar moieties or glycans to different types of molecules, including proteins, lipids, and nucleic acids. Among these, protein glycosylation is one of the most prevalent forms of post-translational modification, playing a crucial role in biological complexity. With more than ten monosaccharides identified within mammalian brain cells and more than 1 × 10¹² possible combinations, the heterogeneity of glycosylation is extensive (Conroy et al., 2021). The diversity of glycans and the complexity of their structures allow for a wide range of protein functions. N-glycans are one of the most abundant forms of glycans and are involved in various cellular functions. N-glycans can be added to proteins at specific sequons, Asn-X-Ser/Thr, and are classified into three main types in mature glycoproteins: high mannose, complex, and hybrid. High mannose N-glycans consist of 5–9 mannose residues linked to a chitobiose core and undergo processing into complex or hybrid forms in the Golgi apparatus (Varki et al., 2017). Complex N-glycans are more diverse and contain various branched structures such as antennae with fucose, galactose, and sialic acid residues. Hybrid N-glycans contain one or more complex branches in conjunction with an oligomannose branch (Fisher and Ungar, 2016). Understanding the specific functions of these different types of N-glycans in protein regulation, folding, and function is an active area of research in the life sciences, including glycobiology.

In particular, brains are the most complex organ and glycosylation is essential for proper brain function, with an estimated 70% or more of brain proteins being glycosylated (Tena and Lebrilla, 2021). Glycosylation is especially important in the development and functioning of the nervous system (Ji et al., 2015; Klaric et al., 2021; Sytnyk et al., 2021; Williams et al., 2022). Furthermore, abnormalities in glycosylation have been implicated in a range of neurological disorders, including epilepsy, schizophrenia, and Alzheimer’s disease (Klaric and Lauc, 2022). Therefore, the significance of glycosylation in brain function is currently a topic of vigorous research, and additional investigations are required to completely comprehend its mechanisms and potential impact on human health (Conroy et al., 2021). While there is emerging evidence supporting the role of N-glycosylation in various brain functions, ranging from normal physiology to pathophysiology, there is still a lack of understanding regarding the spatial and temporal diversity of individual glycans in the brain across different species. Sugars, along with other biomolecules such as amino acids, nucleotides, and lipids, are essential building blocks of life and likely existed in the earliest living organisms. Although previous studies have suggested that sugar structures were conserved across species, unique features of brain N-glycans among mice, rats, macaque monkeys, chimpanzees, and humans have recently been demonstrated with a state-of-the-art technique based on liquid chromatography-mass spectrometry (Lee et al, 2020; Klaric et al., 2023). These two comprehensive studies provide valuable insights into common and distinct glycan structures across species and age groups, which can aid in our understanding of their roles in development and function.

Brain N-glycans across species: Lee et al. (2020) conducted a comprehensive analysis of N-glycans in the human and mouse brain using porous graphitized carbon nano-LC/Q-TOF/TOF-MS/MS. Particularly, the authors compared the prefrontal cortex (PFC) of both species (Figure 1). The study revealed a total of 142 and 136 glycans in human and mouse PFC, respectively. Importantly, all 136 glycans observed in the mouse PFC were also detected in human PFC, indicating that N-glycosylation is highly conserved between these two species. Furthermore, the authors performed quantitative comparisons of the glycan classes in the human and mouse brain, as well as in human and mouse serum, by categorizing glycans into five typical classes: high-mannose, complex or hybrid (C/H), complex/hybrid-fucosylated (C/H-F), complex/hybrid-sialylated (C/H-S), and complex/hybrid-fucosylated and sialylated (C/H-FS). The results showed a high degree of similarity in glycan profiles between human and mouse brains, while significant differences were observed between the respective serum samples. Particularly, higher levels of fucosylation, especially trifucosylation, and lower levels of sialylation were observed in the brain compared to serum. Interestingly, both species exhibited high content of high-mannose glycans in the brain. Given that the mouse is widely used as a model animal for the study of brain functions and disorders, these findings suggest that the mouse is a valuable model system for studying human brain glycosylation and brain function. However, it is important to note that the study also identified unique N-glycans that were observed in human PFC, such as highly branched and fucosylated glycans, as well as fucosylated glycans containing HNK-1 [HSO3-GlcA–Gal–GlcNAc]. These observations indicate that differences exist between the human and mouse brain and further investigation is needed to determine the role of these specific human N-glycans in brain function and development.

Klaric et al. (2023) conducted a comprehensive analysis of N-glycans in four distinct brain regions (or their corresponding hom*ologous equivalents) from humans, chimpanzees, rhesus macaque monkeys, and rats. They utilized an ultra-performance liquid chromatography method based on hydrophilic interactions coupled with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometry after labeling with 2-aminobenzamide. The study identified 145 distinct glycan compositions, which make up a total of 287 distinct N-glycan structures across all adult brain regions and species. Additionally, 60 ubiquitous N-glycans were identified, accounting for approximately 75% of the entire brain N-glycome. Interestingly, the N-glycan profiles were generally similar and conformed to the same overall “brain N-glycosylation template”, characterized by an abundance of high mannose (33.7–44.0%) and hybrid structures (5.9–11.5%), with the remainder being complex N-glycans (41.0–53.7%) and a minor proportion of truncated (< 6%) and paucimannose (< 3%) N-glycans. Although the number of ubiquitous N-glycans identified in the study is relatively low, they actually comprise the bulk of the brain N-glycome, and the overall “brain N-glycosylation template” is consistent across different species. This indicates that the glycan profiles of the brain are well conserved, despite differences in the cytoarchitectural characteristics of brain regions among the species included in the analysis. Despite using different ionization methods (electrospray ionization and MALDI, respectively) and comparing different species (one study compared mouse and human, the other included rat, monkey, chimpanzee, and human), both studies yielded similar results. Specifically, they found that N-glycome profiles were similar across species, indicating that there is a conserved pattern of N-glycosylation in the brain (Lee et al., 2021; Klaric et al., 2023). However, Klaric et al. (2023) have also suggested a unique feature of N-glycan profiles in human brains, namely the prevalence of α(2-6)-linked Neu5Ac among sialyated complex N-glycans. Altogether, both studies indicate that N-glycan profiles are highly conserved across various species. Nonetheless, they also discovered distinct characteristics of N-glycans in the human brain, which may play a critical role in human brain development and higher cognitive function.

Brain N-glycans in development: Lee et al. (2020) conducted a study to investigate developmental changes in N-glycan profiles in the mouse and human PFC (Figure 1). The study analyzed six different age groups of mice and seven different age groups of humans. The results showed that both human and mouse brains had clear separation into two different clusters based on age groups: a young cluster (neonate, infant, toddler, and school age for humans and 1, 2, and 3 weeks old for mice) and an older cluster (teenager, young adult, and adult for humans and 6, 10, and 43 weeks old for mice). These findings may imply that changes in N-glycans of PFC play an important role in brain development and aging and may also have value as a biomarker. The study further identified 24 and 36 glycans that significantly changed between the younger and older clusters in humans and mice, respectively. Among them, three N-glycans were significantly increased and two N-glycans were significantly decreased in aged groups in both humans and mice, suggesting that there might be specific and conserved glycans serving a role in PFC brain development and aging across different species. In addition, one notable discovery is that, among numerical variables such as pH, postmortem interval, sex, race, or cause of death, N-glycans in the PFC were most strongly linked to age in humans. This suggests that studies are needed to dissect the specific role of these glycans in the aging process.

Klaric et al. (2023) examined the profiles of N-glycans in four brain regions (dorsolateral prefrontal cortex, hippocampus, striatum, and cerebellar cortex) across four different species. Additionally, the study investigated the developmental pathways determining the adult brain N-glycome by analyzing corresponding brain regions in the prenatal human brain. The results showed that the global maturation of the brain N-glycome is characterized by an increase in the proportion of hybrid and truncated complex N-glycans, predominantly those that are sialylated. Specifically, the proportion of sialylated N-glycans is lower in the mature brain compared to the developing brain, but sialylated N-glycans tend to bear a higher number of Neu5Ac residues. In addition, a tendency towards elevated utilization of α(2-3)-linked Neu5Ac is observed, with the highest occurrence observed in the dorsolateral prefrontal cortex and striatum. The proportion of high mannose N-glycans remained relatively stable across development throughout all brain regions, though they observed a slight redistribution of the major high mannose subtypes with a slightly lower proportion of Man 9 in adults. Lee et al. (2020) also found specific N-glycans like fucosylated glycans containing sialylated LacdiNAc in a younger cluster (Hex3HexNAc6Fuc1NeuAc2 and Hex4HexNAc5Fuc2NeuAc1). Therefore, consistent with other studies that suggest the significance of sialylated N-glycans during development (Torii et al., 2014; Handa-Narumi et al., 2018), both studies suggest that the regulation of N-glycan sialylation undergoes extensive changes during neurodevelopment in the human brain.

The authors of these studies aimed to investigate variation in N-glycosylation within a species across different regions of the brain, in order to gain insight into the relationship between brain development, functionality, and N-glycosylation. Lee et al. (2020) found that different brain regions in mice exhibited highly distinct profiles of N-glycans, with the exception of the cerebellum, which showed a significant overlap with other brain regions. In contrast, Klaric et al. (2023) performed a comparative analysis of N-glycans across four brain regions in different species and found that the cerebellum had the most distinct N-glycan profiles, with an increased abundance of the Man 6. The discrepancy between these two studies is likely due to differences in the brain regions examined. Lee et al. (2020) analyzed nine different brain regions, including the cerebral cortex, prefrontal cortex, striatum, hippocampus, olfactory bulb, diencephalon, midbrain, pons-medullar, and cerebellum, while Klaric et al. (2023) examined four regions, including the dorsolateral prefrontal cortex, hippocampus, striatum, and cerebellar cortex. Additionally, Lee et al. (2020) represented relatively high levels of Man 6 in both the cerebellum and olfactory bulb, whereas Klaric et al. (2023) did not examine the olfactory bulb. Therefore, the different results between these two studies may be attributed to differences in the brain regions analyzed. Upon closer examination of the N-glycans in the cerebellum by Lee et al. (2020), a unique pattern of N-glycans was identified in a particular cluster (referred to as cluster 1 in the paper), where glycans with high abundance were sialylated, indicating a distinctive feature of the cerebellum. Despite potential differences in glycan profiles due to sample differences (mouse vs. other species and nine brain regions vs. four brain regions), distinct features of cerebellum glycosylation suggest the need for further investigation into the function of specific glycosylation-related enzymes or glycans in cerebellum function.

Conclusion: Glycans constitute a very small proportion of the total molecular weight of the brain but play a crucial role in neurological function. Due to their diversity and modifiability, they can adopt an almost limitless range of structures and functions. The brain, with its unique organization into distinct regions, each with specific functions, is the most complex organ in the body. Therefore, studying glycan structures of the brain has been challenging due to their complexity and the limitations of available analytical techniques. Recent technological advances have enabled more detailed analyses of brain glycans, leading to the creation of glycan libraries from various brain regions and species, ranging from mice to humans. Based on the evidence outlined above, it is now clear that specific N-glycans exhibit distinct expression patterns in different brain regions or specific brains in both humans and other species. The availability of these glycan structures and databases provides a fundamental resource for future studies aimed at understanding brain functions. These libraries also open up new avenues for research, allowing scientists to investigate the specific roles of glycans in neurological diseases, including neurodegenerative disorders such as Alzheimer’s and Parkinson’s, and neuropsychiatric disorders such as depression and schizophrenia. Functional studies utilizing glycan imaging tools, antibodies targeting specific glycans, and techniques to modulate the expression of glycosylation enzymes will be crucial for gaining a deeper understanding of the roles played by specific glycans. Additionally, further investigation is necessary to determine the potential contributions of other types of glycans, including O-glycans and glycolipids, in conjunction with N-glycans. Understanding the various glycosylation patterns associated with brain disorders could offer essential insights into their underlying mechanisms, leading to the development of innovative diagnostic and therapeutic approaches.

This work was supported by the Institute for Basic Science (IBS-R001-D2-2022-A03).

Additional file:Open peer review reports 1 and 2.

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